Alzheimer’s disease brain endothelial-like cells reveal differential drug transporter expression and modulation by potentially therapeutic focused ultrasound

doi:10.21203/rs.3.rs-2605800/v1

Download PDF

Research Article

Alzheimer’s disease brain endothelial-like cells reveal differential drug transporter expression and modulation by potentially therapeutic focused ultrasound

https://doi.org/10.21203/rs.3.rs-2605800/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

The blood-brain barrier (BBB) has a major role in maintaining homeostasis of the brain. The primary function of the BBB is regulating the entry of molecules from the blood to the brain while protecting the brain from potentially harmful endogenous and exogenous substances. Transporters expressed in brain endothelial cells (BECs) mediate the uptake or efflux of compounds to and from the brain and they can also challenge the delivery of drugs into the brain for the treatment of brain disorders including Alzheimer’s disease (AD). AD is the most common form of dementia, however, currently there is limited understanding of the role of BBB transporters in AD-related BBB dysfunction.

Methods

We investigated differences in BBB transporter expression and activity in brain endothelial-like cells (iBECs) differentiated from induced pluripotent stem cells (iPSCs) obtained from people carrying the familial AD Presenilin 1 (PSEN1) mutation (PSEN1 exon 9 deletion; AD-iBECs), healthy controls (Ctrl-iBECs), and isogenic PSEN1 exon 9-corrected lines (PSEN1^COR-iBECs). We first characterized the expression levels of 12 BBB transporters in AD-, Ctrl-, and isogenic iBECs to identify any AD specific differences. We then exposed the cells to focused ultrasound, in the absence (FUS^only) or presence of microbubbles (FUS^+MB), to examine whether the expression or activity of key transporters can be modulated by therapeutic ultrasound, a novel technique allowing for transient BBB opening.

Results

Our results identified differences in the expression of BBB transporters between AD-iBECs and control iBECs, suggesting disease-specific effects on transporter expression. Additionally, our results demonstrated FUS^only and FUS^+MB have the potential to modulate efflux transporter activity. Interestingly, AD-iBECs demonstrated significantly reduced PGP-mediated Aβ accumulation following FUS^+MB, an effect not seen in Ctrl-iBECs, suggesting disease-related differences.

Conclusions

Our findings demonstrate that PSEN1 mutant AD-iBECs possess phenotypical differences compared to PSEN1 corrected and unrelated control iBECs in BBB transporter expression and function. Additionally, we show that FUS^only and FUS^+MB can modulate the BBB transporter expression and functional activity in iBECs, having potential implications on drug penetration and amyloid clearance. These findings highlight the differential responses of patient cells to FUS treatment, with patient-derived models likely providing an important tool for modelling of therapeutic effects of FUS.

Alzheimer’s disease

blood-brain barrier

induced pluripotent stem cells

induced brain endothelial-like cells

transporters

focused ultrasound

ultrasound therapy

drug transport

amyloid-β accumulation

Alzheimer’s disease (AD) is the most common cause of dementia worldwide, with prevalence and incidence increasing with age (1). Although most cases of AD develop symptoms after the age of 65 (known as late-onset AD or sporadic AD), approximately 5% of AD patients develop symptoms before the age of 65 and this is described as early onset AD, also known as a type of familial AD (FAD) (2). FAD is caused by known mutations and cases appear to be inherited in an autosomal dominant manner. Mutations in three genes have been reported to play a role in FAD, including presenilin 1 (PSEN1), presenilin 2 (PSEN2) and amyloid precursor protein (APP) (3).

AD is a neurodegenerative disease which is largely characterised by two neuropathological hallmarks: (1) extracellular accumulation of amyloid-β (Aβ) peptide and (2) intracellular accumulation of hyperphosphorylated tau protein which subsequently forms tau tangles, leading to loss of neurons and neuronal death (4). In addition, emerging evidence indicates that patients with FAD and sporadic AD have a dysfunctional blood-brain barrier (BBB) (5, 6).

The BBB is a physical, metabolic, and immunological barrier that plays a role in maintaining homeostasis in the brain (7). Its main roles are to supply nutrients to, and exclude waste products from the brain and regulate the passage of cells and molecules from the systemic circulation to the central nervous system (CNS) to maintain a highly controlled microenvironment for neuronal function. The BBB is primarily composed of brain endothelial cells (BECs), astrocytes, and pericytes, with BBB cells also interacting with neurons and microglia to form the neurovascular unit (NVU) (8). BECs are tightly connected to each other via tight junctions (TJs) and adherens junctions (AJs), including Occludin, Claudin-5, zonula occludens-1 (ZO-1) and VE-Cadherin, respectively (9). TJs and AJs together control the permeability of the BBB and regulate the passive entry of ions, molecules, and nutrients from the blood to the brain, while protecting the brain from potentially harmful endogenous and exogenous substances, infiltrating immune cells and microorganisms (10). Drug transport into the brain is also selectively controlled by BECs (4, 11, 12)

Moreover, the exchange of molecules between the brain and the blood is controlled by BBB transporters expressed in BECs (12–15). At the BBB multiple different transporter families are expressed. The function of transporters can broadly be divided by pumping molecules “in or out” of the brain, and these are expressed in each of the cellular components of the BBB (16). BBB transporters include members of ATP-binding cassette (ABC) superfamily and the solute carrier (SLC) superfamily (17, 18). While the ABC transporters, which use the energy from ATP hydrolysis, function mostly as efflux transporters, the SLC transporters play a crucial role in transporting small molecules from the blood into the brain (17). Importantly, some BBB transporters are associated with AD and BBB dysfunction. For example, P-glycoprotein (PGP), which is a member of the ABC transporter superfamily, has been reported to play a major role in the clearance of Aβ from the brain in mouse models (19) and the dysregulation of this efflux transporter might be associated with AD pathogenesis (20). Moreover, Multidrug resistance protein 1 (MRP1), another member of the ABC transporter family, is expressed in the apical side of BECs and it plays an important role in mediating inflammatory responses and acts as a defence against the accumulation of reactive oxygen species (ROS) in the brain by pumping out toxins (e.g., glutathione complexes), consequently protecting BBB function (21). In addition, MRP1 plays a crucial role in regulating the efflux of Aβ peptides from the brain to blood (22). Likewise, the BBB restricts > 98% of small drugs and almost 100% of large drugs from entering the brain (23, 24), with BBB transporters playing a crucial role in drug restriction (25). Thus, the BBB and its transporters form a major impediment for researchers developing drugs that target the brain for the treatment of CNS disorders, such as AD (26). Moreover, dysfunction of the BBB associated with AD likely further hinders drug delivery due to the altered physiological conditions of the BBB and the brain (27), which can lead to altered drug metabolism. However, the mechanisms underlying BBB dysfunction in relation to transporter function are yet to be fully investigated in the human (4, 17, 28).

Focused ultrasound (FUS) together with gas-filled microbubbles (FUS^+ MB) has been shown to temporarily open the BBB and increase therapeutic permeability into the brain in both in vitro and in vivo studies (29–34). This method has been shown to be safe in animals and a limited number of human studies and importantly, it has been shown to aid in drug delivery (34, 35) and Aβ clearance in AD mouse models (36–38). On the other hand, FUS without MBs (FUS^only) has been shown to improve memory, cognitive and motor functions and mitigate AD pathogeneses while being safe and efficient as shown in animals and early stage clinical trials in patients (38, 39). Although the effects of FUS^only and FUS^+ MB have been shown to be promising in modulating the brain microenvironment and drug delivery, respectively, their effects on BBB transporters, particularly in the context of AD and in patient-derived models, have not been investigated.

Since changes in BBB transporter expression and function in AD have not been extensively investigated, we aimed to address this in the present study. Here we utilized a patient-derived FAD iBEC model, which we have previously reported to exhibit characteristics of AD (31)). We first investigated differences in BBB transporter expression in AD versus control cells to gain insight into possible dysfunction in BBB transporters in AD. Then, we investigated the effects of FUS^only and FUS^+ MB on key BBB transporter expression as well as the activity of two key BBB efflux transporters, PGP and MRP1. Finally, we investigated the effects of FUS^only and FUS^+ MB on PGP-mediated Aβ uptake. Our results identified key differences in transporter expression and activity between FAD and control iBECs, and we also show that the expression of some transporters is altered following FUS treatments depending on the disease status. Overall, this study provides new insights into BBB transporter changes in FAD and reveals potential modulatory effects of FUS on BBB transporter activity.

Study design

The aim of this research study was to investigate the effects of FAD on BBB transporters and the effect of FUS on transporter expression and function using an in vitro BBB model. To investigate this, we used a patient-derived iPSCs to derive brain endothelial-like cells. In this study a total of six hiPSC lines were used, including two unrelated healthy control lines, two FAD lines harbouring the PSEN1 exon 9 deletion and two analogous isogenic PSEN1 exon 9-corrected controls lines (31, 40). The cells were generated in a controlled laboratory environment and further exposed to FUS treatments, and their responses were investigated in two different timepoints (immediate and 24 h timepoint). Additionally, since the effects of the FUS treatments on BBB transporter expression are unknown, we investigated BBB expression and activity using immunofluorescence, RT-qPCR and functional assays following FUS exposure. The samples sizes were selected based on previous experiments performed by our group and based on previous studies in the field.

Hipsc Expansion And Ibec Differentiation

All hiPSC lines were expanded on human recombinant vitronectin coated culture plates in StemFlex medium (Thermo Fisher), as previously described (31). All the lines studied were successfully characterised using pluripotency markers nanog and SOX2 (Fig. S1). In addition, all lines exhibited a normal karyotype and tested negative for mycoplasma (Table S1). hiPSCs were induced to differentiate into iBECs as previously described (40, 41), and optimised by us (31). Briefly, high quality hiPSCs were detached and singularized using Accutase (Thermo Fisher) and re-plated at 2.0 x10⁵ – 2.5 x10⁵ cells/well in a 6-well plate, pre-coated with Matrigel (Corning) in StemFlex + 10 µM Y27632 (ROCKi, Stemcell technologies). After 24 h, the media was changed to growth media without ROCKi and daily media changes were performed until day-1 with StemFlex. On day 0, the media was changed to Unconditioned (UM) media, composed of DMEM/F12 + Glutamax (1X), Knockout serum replacement (20%) + MEM nonessential amino acids (1x) (all from Thermo Fisher) and β- mercaptoethanol (0.1 mM) (Sigma) to start spontaneous differentiation. After this step, media was changed every other day. After 6 days in UM, the media was switched to ECSR1 + FGFb + RA, composing of Endothelial serum-free medium (Thermo Fisher) (1x) + B27 supplement (2%) (Thermo Fisher) + FGF-2 (20 ng/mL) (Peprotech) and Retinoic acid (10 սM) (Sigma). After 2 days in ECSR1 + FGFb + RA (day 8 of differentiation) cells were re-plated and purified. To purify the cells, media was removed, and cells were treated with Accutase for approximately 30 min at 37⁰C to detach the cells. Cells were flushed off the plate using D-PBS in order to break up the clumps, assuring single cell suspension. Cells were centrifuged at 300 x g for 5 minutes and re-suspended into ECSR1 + FGFb + RA. Cell count was then performed, and cells re-plated on Collagen IV (400 µg/mL, Sigma) + Fibronectin (100 µg/mL, Thermo Fisher) coated plates at the desired cell densities as previously reported (Oikari 2020). To confirm the integrity of the iBEC monolayer, TEER was measured. For this, cells were cultured in the upper compartments of 0.4 µm pore polyester Transwell® 6.5mm inserts (Corning), at a density of 3.0 x 10⁵ cells/insert- and trans-endothelial electrical resistance (TEER) was measured using the EVOM3 Volt/Ohm meter. All lines exhibited TEER values of over 2,000-ohm x cm² and expressed BEC markers (occluding, claudin-5, ZO-1 and VE-cadherin) via immunofluorescence, confirming iBEC differentiation (Fig. S2 & S3).

Immunofluorescence For Hipsc And Ibec Characterization

Immunofluorescence was used to detect the expression of pluripotency markers in iPSCs and BBB-specific markers and transporters in iBECs. Briefly, cells were rinsed twice with D-PBS for 5 minutes and either fixed with ice-cold methanol for 5 min on ice or with 4% paraformaldehyde (PFA) at room temperature (RT) for 20 minutes (refer to Table S2, for antibody-specific information). Following fixation, PFA-fixed cells were permeabilized with 0.3% triton-X for 15 minutes. Cells were then blocked with 2% BSA and 2% normal goat serum (Sigma) in D-PBS for 1 h at RT. Primary antibodies were diluted in blocking solution and incubated overnight at 4℃. The following day, cells were washed three times with D-PBS, and incubated with fluorophore-labelled secondary antibodies (goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 594 from Thermo Fisher, all added 1:250) diluted in blocking solution for 1 h at RT, protected from light. Cells were then washed three times with D-PBS, followed by treatment with Hoechst counterstaining (1:5,000), and coverslips were mounted with ProLong Gold Antifade (Thermo Fisher). Images were obtained using a 20 X objective with a Zeiss 780 confocal microscope in Z stack acquisition.

RNA extraction, cDNA synthesis and quantitative real-time PCR.

For gene expression analysis of transporters in iBECs, culture medium was first removed, and cells were rinsed once with D-PBS. Cells were then treated with TRIZOL ^TM reagent (Thermo Fisher) and scraped off the culture plate using a pipette tip. RNA was extracted using the RNA Miniprep kit (Zymo Research) according to the manufacturer’s instructions. After that, the quality and quantity of RNA was measured using a Nanodrop Spectrophotometer and RNA was converted to cDNA using SensiFAST™ cDNA synthesis kit (Bioline). 18S was used as a housekeeping gene to normalize qRT-PCR results. Following the manufacturer’s instructions, cDNA samples were combined with SensiFAST™ SYBR® Lo-ROX (Bioline) and gene-specific primers to form the reaction solution. The qRT-PCR run was performed in triplicate for each sample on QuantStudio™ 5 Real-Time PCR system (Thermo Fisher Scientific). Briefly, the samples were run for 2 min at 95°C followed by 40 cycles of 5 s at 95°C then 30 s at 60°C. The CT values of each gene was normalized to CT values of 18S (ΔCT values). ΔΔCt values were then calculated as 2^(−ΔCt) and presented as ΔΔCt multiplied by 10⁶. Primer sequences from the genes used in this study are presented in supplementary material (Table S3).

Focused Ultrasound Treatment

The effects of focused ultrasound (FUS) with or without microbubbles (MBs) were tested on iBEC transporter expression and function. The experiments were performed 48 h following iBEC purification on collagen IV and fibronectin coating, as previously described by us (31). For immunofluorescence, transporter functional activity and RNA sample collection assays, cells were grown on 24-well plates. For TEER measurement assays cells were grown on Ø 0.4 µm pore Transwell inserts. Cells were tested under three different conditions (1) untreated (UT), (2) focused ultrasound alone (FUS^only), and (3) focused ultrasound with microbubbles (FUS^+ MB). MBs were prepared in-house as previously described (36, 42) and further optimised by our group to use in in vitro patient-derived model (34). MBs were added at 20 µL per well in 24-well plates and 10 µL per well in Transwell inserts as previously optimized by us (31). Finally, cells were exposed to focused ultrasound (Sonic Concepts) using 0.3 MPa peak rarefactional pressure, 50 cycles/burst, burst period 20 ms, 286 kHz center frequency, and a 120 s sonication time as previously described by us (31). For the FUS^only condition, cells were sonicated following the same parameters without MB addition to the wells. Following treatment, cells were analyzed at two time-points: within 1 h after treatment (immediate timepoint) and at 24 h after treatment, to capture iBEC barrier opening and closing, respectively, as previously demonstrated by us and others (31, 43, 44).

Rhodamine accumulation assay to measure PGP activity.

The functional activity of PGP in iBEC monolayers was measured by the intracellular accumulation of fluorescent rhodamine 123, which is a substrate for PGP. The PGP activity assay using rhodamine 123 was performed as previously described (31, 45, 46). Briefly, 48 h following purification on collagen IV and fibronectin, iBECs were first exposed to FUS^only or FUS^+ MB or left untreated and rhodamine 123 uptake was performed immediately (Imm) or 24 h following treatment. For this, cells were first pre-treated with 10 µM of the PGP inhibitor Cyclosporin-A (CsA, Sigma) or left untreated (only treated with Hanks’ balanced salt solution (HBSS)) and incubated at 37°C for 30 min. Cells were then incubated with 10 µM rhodamine 123 with or without CsA in HBSS at 37°C for 2 h. Following incubation, cells were washed with ice-cold D-PBS for 5 minutes and then lysed with RIPA cell lysate buffer (Sigma) for 10 minutes on a microplate shaker. Fluorescence intensity of cell lysates was measured on a plate reader (Biotek Synergy H4 Multi Mode Plate Reader) using the following wavelengths: 485 nm excitation and 530 nm emission. Results were normalized to total protein amount, which was quantitated using a Pierce ^TM BCA protein assay (Thermo Fisher Scientific), according to the manufacturer’s instructions.

In the absence of CsA, rhodamine 123 is expelled from the cells via the efflux function of PGP, however, when PGP is inhibited by CsA, intracellular accumulation of rhodamine 123 occurs. Thus, elevated intracellular accumulation of rhodamine 123 following PGP inhibition by CsA, equates to higher activity of PGP. To confirm active PGP function, the fold change of rhodamine 123 uptake in + CsA treated cells to non-CsA treated cells for each group (-CsA vs + CsA) was calculated making sure that the accumulation of rhodamine was at least 30% higher in + CsA cells compared to -CsA cells in the UT conditions (45) (Fig. S5A). Following confirmation of PGP activity, the effects of FUS^only and/or FUS^+ MB treatment on rhodamine 123 accumulation (PGP activity) was then measured by calculating fold change in rhodamine 123 accumulation compared to UT in + CsA treated cells at the immediate and 24 h timepoints.

Calcein AM accumulation assay to measure MRP1 activity.

The functional activity of MRP1 in iBEC monolayers was measured by the intracellular accumulation of fluorescent calcein-AM, which is a substrate for MRP1. The MRP1 activity assay using calcein AM was performed as previously described (45, 46). Briefly, 48 h following purification on collagen IV and fibronectin, cells were first exposed to FUS^only or FUS^+ MB or left untreated and calcein-AM uptake was performed immediately (Imm) or 24 h following treatment. For this, cells were first pre-treated with 12.5 nM MK-571 (Sigma), which is an MRP1 inhibitor, or left untreated (treated only with DMEM/F12) and then incubated at 37°C for 30 min. Cells were then treated with or without MK-571 along with 5 nM calcein-AM (Sigma) diluted in DMEM/F12 for 1 h at 37°C. Following incubation, cells were washed with ice-cold D-PBS for 5 min and then lysed with RIPA cell lysate buffer (Sigma) for 10 min on a microplate shaker. Fluorescence intensity of cell lysates was measured on a plate reader (Biotek Synergy H4 Multi Mode Plate Reader), with the following wavelengths: 496 nm excitation and 560 nm emission. First, the fluorescence values were normalized to total protein obtained by BCA assay (as previously described by (Stebbins et al. 2016)). Then, the fold change of + MK-571 treated samples to non-MK-571 samples was analysed using the UT Imm samples ensuring that the accumulation of calcein-AM was in a range of (37% -100%) (Fig. S5B), indicating the presence of MRP1 activity, as previously reported (47). After this, cells that had been exposed to MK-571 were used to examine the effects of FUS^only and FUS^+ MB treatment on calcein AM accumulation at the immediate and 24 h timepoints.

Analysis of PGP-mediated intracellular amyloid-Aβ accumulation.

To measure PGP-mediated the uptake of Aβ, iBECs were exposed to the PGP inhibitor CsA. Briefly, 48 h following purification on collagen IV and fibronectin, iBECs were exposed to FUS^only or FUS^+ MB or left untreated. Then immediately or 24 h following treatment, cells were incubated with 10 µM CsA (Sigma) or without PGP inhibitor (HBSS only) for 1 h at 37°C. Following CsA incubation, cells were then incubated with 5 µM of FITC-conjugated Aβ₄₂ (Bachem) diluted in HBSS for 1 h 30 min at 37°C. After this, cells were washed with ice-cold D-PBS for 5 min and then lysed with RIPA cell lysate buffer (Sigma) for 10 min on a microplate shaker. Fluorescence intensity of cell lysates was measured on a plate reader (Biotek Synergy H4 Multi Mode Plate Reader), as follows: 475 nm excitation and 650 nm emission for Aβ assay. As with the rhodamine 123 and calcein-AM uptake assays, fluorescence values of Aβ accumulation were first normalized to total protein and then effects of FUS^only and FUS^+ MB compared to UT on Aβ accumulation within + CsA treated cells was analyzed as described above (Fig.S5(G-I)).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 9.4.0. Data were analyzed using Student t- test and one-way ANOVA, and the specific multiple comparison tests are described in each figure legend. Values are shown as mean ± SEM. N = biological replicates and n = independent replicates used for each experiment are defined in each figure legend.

Comparison of BBB transporter expression in control and FAD iBECs revealed PSEN1 and non-PSEN1 related changes.

As the role of BBB transporters in AD is poorly understood, we first compared the mRNA levels of selected BBB transporters between iBECs containing PSEN1 FAD mutation (AD), isogenic PSEN1-corrected (PSEN1^COR) and unrelated healthy controls (Ctrl). These included 12 highly expressed BBB transporters. The selected BBB transporters investigated in this study included both ABC and SLC superfamily transporters as well as the low-density lipoprotein receptor- related protein-1 (LRP1) which is a member of receptor-mediated transcytosis (RMT) transporter family, with all selected transporters used in this study being highly expressed in the brain, and some also being associated with BBB function, dysfunction and/or AD pathophysiology (summarized in Table 1).

Table 1

Cell location, function and association with AD of BBB transporters
TRANSPORTERS	LOCATION	FUNCTION & REF.
ABCA1/Cholesterol Transporter	Extensively expressed in brain tissues	Regulates the efflux of cholesterol and phospholipids to APOE’s lipidation, recent in vitro studies in human model of the BBB demonstrated that the dysregulation of cholesterol affects Aβ exchange (48). Downregulates the influx of Aβ across the BBB (49).
ABCB1/ P – Glycoprotein (PGP)	Expressed in BECs, pericytes, astrocytes and neurons	Downregulated at the BBB during normal aging process (50) . Role in the clearance of Aβ from the brain into blood. Transports xenobiotics across the BBB (17)
ABCC1/Multidrug Resistance Protein 1 (MRP1)	Expressed in BECs, astrocytes and pericytes	Low levels of ABCC1 may increase the levels of Aβ40 and Aβ42 in the brain of transgenic mice (51).
ABCC2/Multidrug Resistance Protein 2 (MRP2)	Expressed in BECs.	Upregulated in Alzheimer's models (human and animal) (28).
ABCG2/Breast Cancer Resistant Protein (BCRP)	Overexpressed in BECs	Major role in mediating the efflux of Aβ in BECs(52)
ABCG4/ATP binding cassette subfamily G member 4	Expressed in glial cells, neurons and BECs.	Regulates the efflux of cholesterol (53). Altered ABCG4 leads to increases in Aβ secretion (54).
SLC2A1/GLUT1 = Glucose Transporter 1	Expressed in BECs, neurons, astrocytes and microglia	Role in glucose homeostasis, downregulation of this transporter accelerates BBB disruption, via tight junctions’ protein degradation. (55)
SLC22A3 /OCT3- Solute Carrier Family 22 Member 3	Highly expressed in BECs	Role in the uptake of organic anions/cations into the brain.(56, 57)
SLC22A8/ OAT3 – Solute Carrier Family 22 Member	Expressed in the brain	Role in the efflux transport (brain to blood) of therapeutic agents (e.g., antivirals and antibiotics) (58). It has found to be dysregulated in AD brains and preclinical models of AD, which suggest that might play a role in AD pathogenesis (59)
SLC7A5/ LAT1 – L-type amino acid transporter 1	Highly expressed in the brain	Role in the uptake of essential amino acids and drugs into the BBB (60, 61).
SLCO1A2/OATP12/ Organic anion transporting polypeptide	Highly expressed in the brains and brain regions	The most important SLCO in human brain due to its high expression levels in the brain. Additionally, it plays a role in the uptake of drugs. Alterations in this gene increase the risk of AD, and it has been associated with cortical Aβ deposition in AD (62)
LRP1 – low-density lipoprotein receptor- related protein-1	Extensively expressed in brain tissues	Plays a major role in regulating Aβ levels in the brain, LRP1 expression is reduced with age and in AD patients (63). LRP1 has been shown to play a role in controlling the levels of tau in the brain., and downregulation of LRP1 in in vivo animal models of AD, was shown to reduce the tau spread in the brain (64).

Data from Ctrl-, PSEN1^COR− and AD-iBEC lines were compared for differences in the expression of the selected transporters. We found no differences in the expression of SLC22A8 (OAT3) between the iBEC groups, however, interestingly, significant differences in expression were identified in all the other examined transporter genes (Fig. 1). Of the ATP-dependent transporters, ABCB1 (PGP) (Fig. 1B), ABCC1 (MRP1) (Fig. 1C), ABCC2 (MRP2) (Fig. 1D) and ABCG2 (BCRP) (Fig. 1E) were all significantly downregulated in PSEN1^COR-iBECs and AD-iBECs compared to Ctrl-iBECs. In contrast, ABCA1 was significantly downregulated in AD-iBECs compared to both Ctrl-iBECs and PSEN1^COR-iBECs, while no significant changes were observed in PSEN1^COR-iBECs compared to Ctrl-iBECs (Fig. 1A). When the SLC transporters were examined, SLC2A1 (GLUT1) (Fig. 1G) was significantly downregulated in PSEN1^COR-iBECs compared to Ctrl-iBECs, however in AD-iBECs SLC2A1 was up-regulated against PSEN1^COR-iBECs, but unchanged against Ctrl-iBECs (Fig. 1G). SLC22A3 (OCT3) was downregulated in both PSEN1^COR-iBECs and AD-iBECs compared to Ctrl-iBECs (Fig. 1H). Interestingly, SLCO1A2 (OATP12) was upregulated in PSEN1^COR-iBECs compared to Ctrl-iBECs but downregulated in AD-IBECs compared to PSEN1^COR-iBECs (Fig. 1K). Additionally, the levels of SLC7A5 (LAT1) were downregulated in AD-iBECs compared to PSEN1^COR-iBECs (Fig. 1J). From RMT transporter members, we analyzed the levels of LRP1 which was downregulated in PSEN1^COR-iBECs and AD-iBECs when compared to Ctrl-iBECs (Fig. 1L). The findings are summarized in Table S4.

Effects of FUS treatment on gene expression of ATP-dependent and SLC BBB transporters in control and AD iBECs.

FUS^+ MB is a relatively new technology that can transiently and safely open the BBB to enable drug delivery in in vivo and in vitro studies (31, 38),. In addition, FUS^only without MBs has been shown to have potentially promising effects on brain cell function (65–67). However, whether FUS^only or FUS^+ MB elicits any modulatory effects on BBB transporters has not been previously investigated. Thus, to assess the effects of FUS^only or FUS^+ MB treatments on BBB drug transporter expression, we next analyzed the gene expression of selected transporters in Ctrl-, PSEN1^COR and AD-iBECs at two different timepoints (immediately and at 24 h) following FUS^only and FUS^+ MB; and compared the results to UT samples (31). We then selected three ATP- dependent and three SLC transporters that were found to be significantly different in AD-iBECs compared to Ctrl-iBECs or PSEN1^COR -iBECs to measure how FUS might have a modulation effect in their expression.

The results revealed that, in Ctrl-iBECs the expression of ABCA1 was significantly upregulated immediately after FUS^+ MB treatment compared to UT (Fig. 2A), interestingly this change was not observed 24 h after treatment (Fig. 2A). In PSEN1^COR-iBECs, the expression of ABCA1 was downregulated immediately after FUS^only and FUS^+ MB treatments compared to UT (Fig. 2A), however after 24 h no changes were found between the treatment conditions (Fig. 2A). In contrast, AD-iBECs revealed no changes in the expression of ABCA1 immediately after FUS^only treatment (Fig. 2A). Interestingly, 24 h after FUS^only treatment, we observed a significant downregulation in the expression of ABCA1 compared to UT in AD-iBECs (Fig. 2A).

The relative expression of ABCB1 (PGP) expression was significantly higher in Ctr-iBECs immediately after FUS^+ MB treatment when compared to UT samples (Fig. 2B), however no changes were identified after 24 h (Fig. 2B). In PSEN1^cor-iBECs neither FUS treatments elicited significant changes in ABCB1 expression at each time-points (Fig. 2B). In AD-iBECs, the expression of ABCB1 was significantly increased immediately after FUS^only and FUS^+ MB treatments compared to UT (Fig. 2B), and this increase was also observed 24 h after FUS^+ MB treatment (Fig. 2B).

The relative expression of ABCC1 (MRP1) was not altered in Ctrl-iBECs immediately following FUS^only or FUS^+ MB treatments (Fig. 2C), however, its expression was significantly downregulated at the 24 h time point following FUS^+ MB when compared to UT (Fig. 2C). PSEN1^cor-iBECs and AD-iBECs responded similarly to FUS^only and FUS^+ MB treatments in terms of ABCC1 expression. In both cell groups, the levels of ABCC1 were significantly reduced immediately after FUS^only and FUS^+ MB treatment when compared to UT (Fig. 2C) and 24 h following treatment, ABCC1 was significantly increased following FUS^only treatment when compared to UT (Fig. 2C).

For SLC transporters, the gene expression of SLC2A1 (GLUT1) was upregulated immediately following FUS^+ MB compared to UT in all cell lines analyzed (Ctrl-iBECs, PSEN1^COR-iBECs and AD-iBECs) (Fig. 3A). Interestingly, 24 h after FUS^only treatment in Ctrl-iBECs SLC2A1 was downregulated compared to UT (Fig. 3A) and 24 h after FUS^+ MB treatment in PSEN1^COR-iBECs, SLC2A1 expression was found to be downregulated compared to UT (Fig. 3A). In contrast, no changes were observed in SLC2A1 expression in AD-iBECs samples 24 h following the treatments used in this study (Fig. 3A).

The expression of SLC7A5 in Ctrl-iBECs was downregulated immediately after FUS^+ MB treatment (Fig. 3B), however no changes were observed after 24 h (Fig. 3B). In PSEN1^COR-iBECs, both FUS^only and FUS^+ MB were shown to downregulate the expression of SLC7A5 immediately after treatment when compared to UT (Fig. 3B), however, at 24 h this effect was only seen in the FUS^+ MB condition (Fig. 3B). Interestingly, immediately after FUS^only treatment in AD-iBECs we observed a significant downregulation of SLC7A5 expression when compared to UT (Fig. 3B), however, no changes were observed after 24 h in either treatment condition used in this study (Fig. 3B).

The expression of SLCO1A2 (OATP12), in Ctrl-iBECs was found to be upregulated immediately following FUS^only and FUS^+ MB when compared to UT (Fig. 3C), however, after 24 h following FUS^only and FUS^+ MB the effects were found to be opposite. showing a downregulation in expression when compared to UT (Fig. 3C). In PSEN1^COR-iBECs FUS^only and FUS^+ MB induced a downregulation in SLCO1A2 expression compared to UT immediately after treatment (Fig. 3C), while in 24 h samples SLCO1A2 downregulation was only observed in FUS^+ MB treated samples (Fig. 3C). In contrast, no changes were observed in SLCO1A2 expression following any of the treatments used in this study for AD-iBECs (Fig. 3C). The findings are summarized in Table S4.

Effects Of Fus Treatment On Pgp Activity In Control And Ad-ibecs

As gene expression does not necessarily equate directly to functional activity of drug transporters, we next examined the effects of FUS^only and FUS^+ MB on PGP functional activity in iBECs. To achieve this, we measured the intracellular accumulation of the PGP substrate rhodamine 123 following FUS^only and FUS^+ MB treatment after performing PGP inhibition with CsA, a common assay used to measure PGP activity (31, 45, 68, 69).

Our data revealed that in Ctrl-iBECs no differences in rhodamine 123 accumulation were identified following FUS treatments compared to UT (Fig. 4A & 4B). In contrast, for PSEN1^COR−iBECs the accumulation of rhodamine 123 was significantly increased immediately after FUS^+ MB when compared to UT (Fig. 4C), and after 24 h rhodamine 123 accumulation was significantly decreased following FUS^only when compared to UT (Fig. 4C), suggesting potential modulatory effects on PGP activity. Similar to Ctrl-iBECs, rhodamine 123 accumulation was not altered in AD-iBECs immediately after FUS treatments when compared to UT (Fig. 4E & 4F), but at 24 h, rhodamine 123 accumulation was significantly decreased in FUS^+ MB treated condition compared to UT, suggesting increased PGP activity (Fig, 4F).

Effects of FUS treatment on MRP1 activity in control and AD-iBECs.

To assess MRP1 activity following FUS^only and FUS^+ MB treatments we used a calcein-AM uptake assay. Calcein-AM is an MRP1 substrate dye that has been extensively used to measure MRP1 activity, together with the MRP1 inhibitor MK-571 (47, 70). Similar to the rhodamine 123 uptake, we measured calcein-AM uptake in the presence of the MRP1 inhibitor MK-571 for all groups (UT, FUS ^only and FUS^+ MB) immediately and 24 h post treatment, respectively.

Our data revealed that calcein-AM uptake was significantly decreased in Ctrl-iBECs immediately after FUS^only and FUS^+ MB treatment compared to UT (Fig. 5A & 5B), suggesting increased MRP1 activity. Reduced calcein-AM uptake following FUS^+ MB was maintained in Ctrl-iBECs at the 24 h timepoint (Fig. 5B). Similar to Ctrl-iBECs, decreased calcein-AM uptake was also observed in PSEN1^COR-iBECs following FUS^only at the immediate timepoint, however, no effects were seen for FUS^+ MB in either timepoint (Fig. 5C & 5D), Interestingly, similar effects were seen for AD-iBECs as for the Ctrl-iBECs, with the accumulation of calcein-AM significantly reduced compared to UT immediately after FUS^only and FUS^+ MB treatments, suggesting a higher activity of MRP1 (Fig. 5E & 5F). However, in contrast to Ctrl-iBECs, calcein-AM uptake was significantly increased 24 h following FUS^+ MB, indicating decreased MRP1 activity at the later timepoint (Fig. 5F).

Effects Of Fus Treatment On Pgp-mediated Aβ Uptake In Control And Ad-ibecs

Finally, we investigated whether FUS^only or FUS^+ MB treatment could modulate PGP-mediated Aβ uptake. PGP has been reported to play a role in Aβ clearance (71) and PGP expression is reported to be altered in AD patients, (26, 72). To confirm that PGP mediates uptake of Aβ in iBECs, we performed PGP inhibition via CsA, which resulted in increased Aβ (Fig. S5C).

When compared to UT we observed no changes in intracellular Aβ accumulation in Ctrl-iBECs following FUS treatments at immediate or 24 h timepoints (Fig. 6A & B). In contrast, in PSEN1^COR-iBECs, Aβ accumulation was significantly increased immediately following FUS^+ MB treatment (Fig. 6C). However, at the 24 h timepoint no significant effects of FUS treatments on Aβ accumulation were identified in PSEN1^COR-iBECs. Interestingly, in AD-iBECs a consistent effect following FUS^+ MB treatment was seen with a significant reduction in Aβ accumulation identified immediately and 24 h following FUS^+ MB treatment compared to UT, suggesting a decrease in PGP-mediated Aβ uptake (Fig. 6E & F).

Transporters at the BBB play a vital role in regulating the entry of molecules from the blood to the brain, but some, such as efflux pumps, challenge the delivery of drugs into the brain (27). In addition, in brain disorders such as AD, transporter activity is altered, potentially contributing to disease progression (73, 74); however the full extent of AD-related changes in BBB transporters in human are not known. As such, the contribution of BBB transporters in AD pathogenesis as well as their effects on drug delivery can be a major impediment to drug therapy. As previously reported by our group (31, 34) and others, BBB opening by FUS^+ MB is a novel technology that is becoming a promising tool to increase drug delivery into the brain (75, 76). In addition, FUS^only can have modulatory effects on the brain that are not well understood (31, 74). However, the molecular effects of FUS^only and FUS^+ MB on human BBB transporters are still unknown with these treatments potentially providing a means to modulate transporter activity. Thus, in this study, we first performed a detailed investigation of the expression of 12 BBB transporters known to be related to AD or BBB dysfunction using our previously reported familial iBEC in vitro model (31). We then used this model to investigate the effects of FUS^only and FUS^+ MB on BBB transporter expression and activity to identify potential novel modulatory effects of therapeutic FUS.

Following confirmation of iBEC lineage differentiation from Ctrl, AD and PSEN1^COR iPSC lines, we first screened the expression of 12 selected transporters (Table.1). Of the studied transporters, our results demonstrated that the relative gene expression of ABCB1 (PGP), ABCC1 (MRP1), ABCC2 (MRP2), ABCG2 (BCRP), ABCG4, SLC22A3 (OCT3) and LRP1 was significantly downregulated in PSEN1^COR and AD-iBECs compared to Ctrl-iBECs suggesting potentially reduced activity in AD patients irrespective of the PSEN1 mutation. Any dysregulation in BBB transporters can affect homeostasis in the brain which could potentially contribute to AD pathogenesis. For instance, the ABCB1 (PGP) is known to be downregulated at the BBB during normal aging process (50) and it has been found to be downregulated in human tissues and animal models of AD (17). PGP also has a reported role in the clearance of Aβ from the brain to the blood, thus a deficit of PGP can lead to Aβ accumulation in CNS (77), highlighting its potential key role in AD pathogenesis. Additionally, a previous study has shown that low levels of ABCC1 (MRP1), might correlate with increased levels of Aβ40 and Aβ42 in the CNS (51), potentially contributing to the formation of Aβ plaques. Thus, the simultaneous downregulation of the two genes ABCB1 (PGP) and ABCC1 (MRP1) as seen in our model, could potentially result in increased Aβ plaque formation, enhancing FAD progression. As such, our results indicate that patient cell-based models can be used to study AD-related BBB dysfunction.

Interestingly ABCA1, SLC7A5 (LAT1) and SLCO1A2 (OATP12) were found to be differentially expressed in AD-iBECs compared to both Ctrl- and PSEN1^COR-iBECs, suggesting their association with the PSEN1 mutation. ABCA1 encodes for a transporter that drives the efflux of cholesterol and directly downregulates the influx of Aβ across the BBB (17, 49, 78). LAT1 in turn plays an important role in the uptake of essential amino acids (60), and in a previous study, LAT1 was not found to be altered in AD patients (in genes encoding APP and PSEN1) (61). SLCO1A2 (OATP12) is an important SLCOs in the human brain, highly expressed in the brain and in specific regions, such as BBB, where it plays a major role in uptake of drugs into the brain (56, 79{Roth, 2012 #131, 80). These findings are also consistent with previous studies which have shown that alterations in this gene might potentially contribute to AD (62). Considering the important functions of ABCA1, LAT1 and OATP12 in maintaining brain homeostasis, investigating their roles at a more in-depth level in relation to AD would be important and could potentially identify new therapeutic targets.

Because BBB-specific transporters provide potential therapeutic targets in AD, there is an interest in identifying whether we can modulate their activity. We have previously reported the ability to model FUS^+ MB-mediated BBB opening using the same patient-derived iBEC model as in the current study (31, 34). We have also previously demonstrated that the mRNA levels of TJPs are altered following FUS^+ MB (31), however, the effect of FUS^only and FUS^+ MB on BBB transporter function remains sparsely uninvestigated. From the 12 BBB transporters we initially screened, we selected three ABC superfamily members and three SLC superfamily that were down- or up-regulated in PSEN1^COR-iBECs and/or AD-iBECs when compared to Ctrl-iBECs to investigate the effects of FUS^only and FUS^+ MB on their gene expression.

The immediate effects of FUS^only and FUS^+ MB on ABCA1 varied between the three studied cell groups, however, interestingly, ABCB1 (PGP) expression was increased following FUS^+ MB in both Ctrl- and AD-iBECs group, suggesting potentially stimulatory effect of FUS^+ MB on PGP gene expression. This effect was seen also 24 h following treatment in AD-iBECs, suggesting potentially slower recovery following FUS^+ MB, which is consistent with our previous study (31). Similar to FUS^+ MB, FUS^only stimulated PGP expression in AD-iBECs, although no effect was seen of this treatment in the other cell groups. Further confirmation for stimulated PGP expression following FUS^+ MB was seen following functional activity assays, which demonstrated increased activity of PGP (indicated by decreased rhodamine 123 uptake) 24 h following FUS^+ MB in AD-iBECs. These findings suggest FUS could potentially be used to stimulate PGP expression, which in turn mediates clearance of Aβ from the brain to the blood (19)

For the relative gene expression of ABCC1 (MRP1), there appeared to be an AD (non PSEN1-related) response to FUS^only and FUS^+ MB as both PSEN1^COR and AD-iBECs demonstrated a strong downregulation in ABCC1 (MRP1) expression immediately following treatments, not observed in Ctrl-iBECs. Similarly, at the 24 h timepoint while Ctrl-iBECs demonstrated significantly reduced ABCC1 (MRP1) expression in FUS^+ MB treated cells, ABCC1 (MRP1) expression was significantly increased in FUS^only treated PSEN1^COR and AD-iBECs when compared to UT. These results indicate a potential patient-specific response to FUS^only and FUS^+ MB and that some transporters might be differentially altered following FUS treatments depending on the disease status. The strong downregulation in ABCC1 (MRP1) expression following FUS treatments in PSEN1^COR and AD-iBECs also suggests that there is a potential to downregulate ABCC1 (MRP1) gene expression during a short window following treatment. In contrast to gene expression results, the functional activity analysis of MRP1 following FUS treatments at the immediate timepoint demonstrated increased MRP1 activity (reduced calcein-AM uptake) in all the cell lines. However, differences were seen at the 24 h timepoint, where increased MRP1 activity was still seen in Ctrl-iBECs, while in AD-iBECs MRP1 activity was decreased (increased calcein-AM uptake) following FUS^+ MB, which correlated with the gene expression results of the immediate timepoint. Overall, our results demonstrate that as with PGP, the effects of FUS^+ MB on MRP1 activity appear to have a longer-lasting effect on AD-iBECs, with effects seen at the 24 h timepoint, not observed in control or corrected iBEC lines. This supports the transient effects of FUS shown by us and others previously and that FUS response may last longer in AD cells compared to the control (31, 34, 81)

Interestingly, SLC2A1 (GLUT1) expression was significantly increased immediately following FUS^+ MB treatment in all the cells groups (Ctrl-iBECs, PSEN1^COR-iBECs and AD-iBECs), suggesting the stimulatory effects of FUS^+ MB on GLUT1. Although at 24 h following FUS treatments the expression of SLC2A1 (GLUT1) was no longer induced, the results suggest that FUS^+ MB might increase the activity of GLUT1 during a short time window, similar to PGP and MRP1, having potential benefits in cases where glucose transport to the brain is impaired (82). FUS treatments appear to mainly have a downregulatory effect on SLC7A5 (LAT1) expression, indicating a potential inhibitory effect of FUS on SLC7A5 (LAT1) expression, and similar to SLC2A1 (GLUT1), the effects of FUS treatments on SLC7A5 (LAT1) expression appear temporary. Interestingly, our results demonstrated that while in Ctrl-iBECs SLCO1A2 (OATP12) expression was initially increased and then decreased following FUS^only and FUS^+ MB, the same effects were not evident in PSEN1^COR and AD-iBECs. PSEN1^COR-iBECs demonstrated a continued decreased SLCO1A2 (OATP12) following FUS^+ MB, while no effect on SLCO1A2 expression following FUS^only treatment was observed. These results suggest that some transporters might be resistant to FUS modulation depending on their expression levels or the disease-status of the patient. The overall lower expression of SLCO1A2 (OATP12) in untreated AD-iBECs compared to untreated Ctrl- and PSEN^COR-iBECs might contribute to the fact that no changes in this transporter were identified following FUS treatments in AD-iBECs. To confirm the functional changes of FUS on SLC transporters, functional assays would be valuable. However, functional assays for SLCs are more complex and are not as well established as efflux transporter assays and were for that reason not explored in this study (83). Together these results corroborate the findings in the field, and reinforces the hypothesis of BBB transporter dysregulation in AD patients (4, 7, 84).

Finally, we used our in-vitro iBEC model to investigate changes in cellular Aβ uptake following FUS^only and FUS^+ MB following PGP inhibition to establish a potential link between FUS-mediated Aβ clearance via PGP. Interestingly, while minimal changes were seen in Ctrl- and PSEN1^COR-iBECs, our results demonstrated significantly decreased cellular Aβ accumulation in AD-iBECs in which PGP had been inhibited via CsA following FUS^+ MB immediately and 24 h following treatment. These results suggest increased PGP-mediated Aβ clearance from the cells which also correlates with the rhodamine 123 uptake results. As such, our data suggests that PGP is likely involved in Aβ clearance, which could potentially be increased with FUS^+ MB, with some disease types potentially more susceptible to these effects. To confirm that the decreased cellular Aβ uptake also correlates with increased Aβ clearance from the brain, cell culture media analysis of soluble Aβ would be needed. However, in our previous study using the same AD-iBECs in a Transwell culture setting, we demonstrated persistent Aβ clearance across the AD-iBEC barrier following FUS^+ MB, supporting the findings of the current study (31).

In conclusion, our findings demonstrate that PSEN1 mutant AD-iBECs possess phenotypical differences compared to PSEN1 corrected and unrelated control iBECs in BBB transporter expression and function, which further supports the contribution of FAD mutations on BBB dysfunction. Additionally, we show for the first time in the human AD BBB cell model that FUS^only and FUS^+ MB can modulate the BBB transporter expression and functional activity in iBECs, having potential implications on drug penetration and amyloid clearance. Our results further highlight the differential responses that patient cells to FUS treatment, with patient-derived models likely providing an important tool for modelling therapeutic responses of FUS.

Aβ – Amyloid- Beta

ABC families – ATP- binding cassette

ABCA1 – Cholesterol Transporter 1

ABCB1 (P-GP) – P-glycoprotein

ABCC1 (MRP1) – Multidrug Resistance Associated Protein 1

ABCC2 (MRP2) – Multidrug Resistance Associated Protein 2

ABCG2 (BCRP) – Breast Cancer Resistant Protein

ABCG4 – ATP Binding Cassette Subfamily G Member 4

AD – Alzheimer’s Disease

AJs – Adherens Junctions

APP – Amyloid Precursor Protein

APOE – Apolipoprotein E

BECs – Brain Endothelial Cells

BBB – Blood-Brain Barrier

BSA – Bovine serum albumin

cDNA – complementary DNA

CNS – Central Nervous System

CsA – Cyclosporin-A

-CsA – non-cyclosporin-A

+CsA – with cyclosporin-A

Ctrl – unrelated healthy controls

DMEM F12 – Dulbecco’s Modified Eagle Medium F12

D-PBS - Dulbecco’s phosphate-buffered saline

ECS – Endothelial Cells

ECSR1 – Endothelial Serum Free Medium

FAD – Familial Alzheimer’s disease

FGFb – Transforming growth factor beta

FUS – Focused Ultrasound

FUS^only– focused ultrasound alone

FUS^+MB – focused ultrasound with microbubbles

HBSS – Hanks’ Balanced Salt Solution

hiPSCs – Human-Induced Pluripotent Stem Cells

iBECs – Induced Brain Endothelial-like Cells

iPSCs – Induced Pluripotent Stem Cells

Imm. – Immediately

LRP1 – low-density lipoprotein receptor-related protein 1

MBs – Microbubbles

MK-571 – Cysteinyl Leukotriene Receptor 1 Antagonist

-MK-571 – non- MK-571

+MK-571 – with MK-571

MRP1 – Multidrug Resistance Associated Protein 1

NVU – Neurovascular Unit

PGP – P – Glycoprotein

PFA – Paraformaldehyde

PSEN1 – Presenilin 1

PSEN1^COR - isogenic PSEN1 exon 9-corrected lines

PSEN2 – Presenilin 2

qPCR – quantitative polymerase chain reaction

RA – Retinoic acid

RNA – Ribonucleic Acid

RMT – Receptor-Mediated- Transcriptions

ROS – Reactive Oxygen Species

ROCKi - ROCK pathway inhibitor

RT – Room Temperature

SLC – Solute Carrier family

SLC2A1 (GLUT1) – Glucose Transporter 1

SLC22A8 (OAT3) – Organic Anion Transporter 3

SLC22A3 (OCT3) – Solute Carrier Family 22 Member A

SLC7A5 (LAT1) – L-type amino acid transporter 1

SLCO1A2 (OATP12) – Organic Anion Transporter Polypeptide

TEER – Transendothelial Electrical Resistance Measurement

TJs – Tight Junctions

TJP – Tight Junctions Proteins

UM – Unconditioned Media

UT – Untreated

ZO-1 – Zonula occludens-1

Ethics approval

This study has been approved by the QIMR Berghofer Medical Research Institute Human Ethics Committee (Tittle: Investigating mechanisms of neurodegeneration and development new therapeutic treatments; Date of approval: 1^st August 2016; P2197).

Consent for publication

Not applicable.

Availability of data and materials

Data used to obtaine the results presented in the manuscript can be obtained from the corresponding author upon reasonable request.

Compenting interests

The authors declare no competing interests.

Funding

This research was suportted by QUT Postgraduate Research Award (QUTPRA) and QUT Higher Degree Research Tuition Fee Scholarship 2020 (to J.C.S.C). This research was supported by QIMR Berghofer SEED grant (to L.E.O), NHMRC Project grants APP1125796 (to A.R.W.). A.R.W. is a recipient of an NHMRC Senior Research Fellowship (APP1118452). This project was supported through the Academy of Finland under the aegis of JPND—www.jpnd.eu––and European Union's Horizon 2020 research and innovation program under grant agreement no. 643417 (to J.K). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Author contributions

J.C.S.C and L.E.O designed the project. J.C.S.C performed most experiments, analysed the data, and wrote the manuscript. J.M.W provided technical assistance with the ultrasound experiments. C.C-L and L.M.R processed samples. S.L provide technical assistance in functional assays and immunofluorescence experiments. J.K provided the iPSC lines used in this study. L.E.O and A.R.W interpreted the data, reviewed and edited the manuscript.

Acknowledgments

We also thank Dr Rucha Pandit and Prof Jürgen Götz, from Queensland Brain Institute (QBI) for their technical assistance in focused ultrasound experiments. We also thank the Microscopy facility at QIMR Berghofer especially Dr Tam Hong Nguyen for technical assistance with the microscopy imaging.

Mendez MF. Early-onset Alzheimer Disease and Its Variants. Continuum (Minneap Minn). 2019;25(1):34-51.
Mendez MF. Early-Onset Alzheimer Disease. Neurol Clin. 2017;35(2):263-81.
Arber C, Toombs J, Lovejoy C, Ryan NS, Paterson RW, Willumsen N, et al. Familial Alzheimer's disease patient-derived neurons reveal distinct mutation-specific effects on amyloid beta. Mol Psychiatry. 2020;25(11):2919-31.
Pereira CD, Martins F, Wiltfang J, da Cruz ESOAB, Rebelo S. ABC Transporters Are Key Players in Alzheimer's Disease. J Alzheimers Dis. 2018;61(2):463-85.
Sweeney MD, Zhao Z, Montagne A, Nelson AR, Zlokovic BV. Blood-Brain Barrier: From Physiology to Disease and Back. Physiol Rev. 2019;99(1):21-78.
Montagne A, Zhao Z, Zlokovic BV. Alzheimer's disease: A matter of blood-brain barrier dysfunction? J Exp Med. 2017;214(11):3151-69.
Barar J, Rafi MA, Pourseif MM, Omidi Y. Blood-brain barrier transport machineries and targeted therapy of brain diseases. Bioimpacts. 2016;6(4):225-48.
Kadry H, Noorani B, Cucullo L. A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity. Fluids Barriers CNS. 2020;17(1):69.
Tietz S, Engelhardt B. Brain barriers: Crosstalk between complex tight junctions and adherens junctions. J Cell Biol. 2015;209(4):493-506.
Kubotera H, Ikeshima-Kataoka H, Hatashita Y, Allegra Mascaro AL, Pavone FS, Inoue T. Astrocytic endfeet re-cover blood vessels after removal by laser ablation. Sci Rep. 2019;9(1):1263.
Redzic Z. Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers: similarities and differences. Fluids Barriers CNS. 2011;8(1):3.
Fricker G, Miller DS. Modulation of drug transporters at the blood-brain barrier. Pharmacology. 2004;70(4):169-76.
Deo AK, Borson S, Link JM, Domino K, Eary JF, Ke B, et al. Activity of P-Glycoprotein, a beta-Amyloid Transporter at the Blood-Brain Barrier, Is Compromised in Patients with Mild Alzheimer Disease. J Nucl Med. 2014;55(7):1106-11.
Sekhar GN, Fleckney AL, Boyanova ST, Rupawala H, Lo R, Wang H, et al. Region-specific blood-brain barrier transporter changes leads to increased sensitivity to amisulpride in Alzheimer's disease. Fluids Barriers CNS. 2019;16(1):38.
Banks WA. Characteristics of compounds that cross the blood-brain barrier. BMC Neurol. 2009;9 Suppl 1:S3.
Urquhart BL, Kim RB. Blood-brain barrier transporters and response to CNS-active drugs. Eur J Clin Pharmacol. 2009;65(11):1063-70.
Jia Y, Wang N, Zhang Y, Xue D, Lou H, Liu X. Alteration in the Function and Expression of SLC and ABC Transporters in the Neurovascular Unit in Alzheimer's Disease and the Clinical Significance. Aging Dis. 2020;11(2):390-404.
Morris ME, Rodriguez-Cruz V, Felmlee MA. SLC and ABC Transporters: Expression, Localization, and Species Differences at the Blood-Brain and the Blood-Cerebrospinal Fluid Barriers. AAPS J. 2017;19(5):1317-31.
Erdo F, Krajcsi P. Age-Related Functional and Expressional Changes in Efflux Pathways at the Blood-Brain Barrier. Front Aging Neurosci. 2019;11:196.
Aryal M, Fischer K, Gentile C, Gitto S, Zhang YZ, McDannold N. Effects on P-Glycoprotein Expression after Blood-Brain Barrier Disruption Using Focused Ultrasound and Microbubbles. PLoS One. 2017;12(1):e0166061.
Gil-Martins E, Barbosa DJ, Silva V, Remiao F, Silva R. Dysfunction of ABC transporters at the blood-brain barrier: Role in neurological disorders. Pharmacol Ther. 2020;213:107554.
Behl T, Kaur I, Sehgal A, Kumar A, Uddin MS, Bungau S. The Interplay of ABC Transporters in Abeta Translocation and Cholesterol Metabolism: Implicating Their Roles in Alzheimer's Disease. Mol Neurobiol. 2021;58(4):1564-82.
Pardridge WM. The blood-brain barrier: bottleneck in brain drug development. NeuroRx. 2005;2(1):3-14.
He Q, Liu J, Liang J, Liu X, Li W, Liu Z, et al. Towards Improvements for Penetrating the Blood-Brain Barrier-Recent Progress from a Material and Pharmaceutical Perspective. Cells. 2018;7(4).
Fricker G. Drug transport across the blood-brain barrier. Ernst Schering Res Found Workshop. 2002(37):139-54.
Cirrito JR, Deane R, Fagan AM, Spinner ML, Parsadanian M, Finn MB, et al. P-glycoprotein deficiency at the blood-brain barrier increases amyloid-beta deposition in an Alzheimer disease mouse model. J Clin Invest. 2005;115(11):3285-90.
Achar A, Myers R, Ghosh C. Drug Delivery Challenges in Brain Disorders across the Blood-Brain Barrier: Novel Methods and Future Considerations for Improved Therapy. Biomedicines. 2021;9(12).
Qosa H, Miller DS, Pasinelli P, Trotti D. Regulation of ABC efflux transporters at blood-brain barrier in health and neurological disorders. Brain Res. 2015;1628(Pt B):298-316.
Burgess A, Shah K, Hough O, Hynynen K. Focused ultrasound-mediated drug delivery through the blood-brain barrier. Expert Rev Neurother. 2015;15(5):477-91.
D'Haese PF, Ranjan M, Song A, Haut MW, Carpenter J, Dieb G, et al. beta-Amyloid Plaque Reduction in the Hippocampus After Focused Ultrasound-Induced Blood-Brain Barrier Opening in Alzheimer's Disease. Front Hum Neurosci. 2020;14:593672.
Oikari LE, Pandit R, Stewart R, Cuni-Lopez C, Quek H, Sutharsan R, et al. Altered Brain Endothelial Cell Phenotype from a Familial Alzheimer Mutation and Its Potential Implications for Amyloid Clearance and Drug Delivery. Stem Cell Reports. 2020;14(5):924-39.
Arvanitis CD, Askoxylakis V, Guo Y, Datta M, Kloepper J, Ferraro GB, et al. Mechanisms of enhanced drug delivery in brain metastases with focused ultrasound-induced blood-tumor barrier disruption. Proc Natl Acad Sci U S A. 2018;115(37):E8717-E26.
Zhang F, Xu CL, Liu CM. Drug delivery strategies to enhance the permeability of the blood-brain barrier for treatment of glioma. Drug Des Devel Ther. 2015;9:2089-100.
Wasielewska JM, Chaves JCS, Johnston RL, Milton LA, Hernandez D, Chen L, et al. A sporadic Alzheimer's blood-brain barrier model for developing ultrasound-mediated delivery of Aducanumab and anti-Tau antibodies. Theranostics. 2022;12(16):6826-47.
Raymond SB, Skoch J, Hynynen K, Bacskai BJ. Multiphoton imaging of ultrasound/Optison mediated cerebrovascular effects in vivo. J Cereb Blood Flow Metab. 2007;27(2):393-403.
Pandit R, Leinenga G, Gotz J. Repeated ultrasound treatment of tau transgenic mice clears neuronal tau by autophagy and improves behavioral functions. Theranostics. 2019;9(13):3754-67.
Leinenga G, Koh WK, Gotz J. A comparative study of the effects of Aducanumab and scanning ultrasound on amyloid plaques and behavior in the APP23 mouse model of Alzheimer disease. Alzheimers Res Ther. 2021;13(1):76.
Liu X, Naomi SSM, Sharon WL, Russell EJ. The Applications of Focused Ultrasound (FUS) in Alzheimer's Disease Treatment: A Systematic Review on Both Animal and Human Studies. Aging Dis. 2021;12(8):1977-2002.
Nicodemus NE, Becerra S, Kuhn TP, Packham HR, Duncan J, Mahdavi K, et al. Focused transcranial ultrasound for treatment of neurodegenerative dementia. Alzheimers Dement (N Y). 2019;5:374-81.
Lippmann ES, Al-Ahmad A, Azarin SM, Palecek SP, Shusta EV. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources. Sci Rep. 2014;4:4160.
Qian T, Maguire SE, Canfield SG, Bao X, Olson WR, Shusta EV, et al. Directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells. Sci Adv. 2017;3(11):e1701679.
Leinenga G, Gotz J. Scanning ultrasound removes amyloid-beta and restores memory in an Alzheimer's disease mouse model. Sci Transl Med. 2015;7(278):278ra33.
Chen L, Sutharsan R, Lee JL, Cruz E, Asnicar B, Palliyaguru T, et al. Claudin-5 binder enhances focused ultrasound-mediated opening in an in vitro blood-brain barrier model. Theranostics. 2022;12(5):1952-70.
Lipsman N, Meng Y, Bethune AJ, Huang Y, Lam B, Masellis M, et al. Blood-brain barrier opening in Alzheimer's disease using MR-guided focused ultrasound. Nat Commun. 2018;9(1):2336.
Stebbins MJ, Wilson HK, Canfield SG, Qian T, Palecek SP, Shusta EV. Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells. Methods. 2016;101:93-102.
Ansbro MR, Shukla S, Ambudkar SV, Yuspa SH, Li L. Screening compounds with a novel high-throughput ABCB1-mediated efflux assay identifies drugs with known therapeutic targets at risk for multidrug resistance interference. PLoS One. 2013;8(4):e60334.
Olson DP, Taylor BJ, Ivy SP. Detection of MRP functional activity: calcein AM but not BCECF AM as a Multidrug Resistance-related Protein (MRP1) substrate. Cytometry. 2001;46(2):105-13.
Kuntz M, Candela P, Saint-Pol J, Lamartiniere Y, Boucau MC, Sevin E, et al. Bexarotene Promotes Cholesterol Efflux and Restricts Apical-to-Basolateral Transport of Amyloid-beta Peptides in an In Vitro Model of the Human Blood-Brain Barrier. J Alzheimers Dis. 2015;48(3):849-62.
Boehm-Cagan A, Bar R, Liraz O, Bielicki JK, Johansson JO, Michaelson DM. ABCA1 Agonist Reverses the ApoE4-Driven Cognitive and Brain Pathologies. J Alzheimers Dis. 2016;54(3):1219-33.
van Assema DM, Lubberink M, Rizzu P, van Swieten JC, Schuit RC, Eriksson J, et al. Blood-brain barrier P-glycoprotein function in healthy subjects and Alzheimer's disease patients: effect of polymorphisms in the ABCB1 gene. EJNMMI Res. 2012;2(1):57.
Krohn M, Lange C, Hofrichter J, Scheffler K, Stenzel J, Steffen J, et al. Cerebral amyloid-beta proteostasis is regulated by the membrane transport protein ABCC1 in mice. J Clin Invest. 2011;121(10):3924-31.
Do TM, Noel-Hudson MS, Ribes S, Besengez C, Smirnova M, Cisternino S, et al. ABCG2- and ABCG4-mediated efflux of amyloid-beta peptide 1-40 at the mouse blood-brain barrier. J Alzheimers Dis. 2012;30(1):155-66.
Dodacki A, Wortman M, Saubamea B, Chasseigneaux S, Nicolic S, Prince N, et al. Expression and function of Abcg4 in the mouse blood-brain barrier: role in restricting the brain entry of amyloid-beta peptide. Sci Rep. 2017;7(1):13393.
Sano O, Tsujita M, Shimizu Y, Kato R, Kobayashi A, Kioka N, et al. ABCG1 and ABCG4 Suppress gamma-Secretase Activity and Amyloid beta Production. PLoS One. 2016;11(5):e0155400.
Liu L, Liu XD. Alterations in function and expression of ABC transporters at blood-brain barrier under diabetes and the clinical significances. Front Pharmacol. 2014;5:273.
Roth M, Obaidat A, Hagenbuch B. OATPs, OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. Br J Pharmacol. 2012;165(5):1260-87.
Koepsell H. General Overview of Organic Cation Transporters in Brain. Handb Exp Pharmacol. 2021;266:1-39.
Deodhar M, Rihani SBA, Darakjian L, Turgeon J, Michaud V. Assessing the Mechanism of Fluoxetine-Mediated CYP2D6 Inhibition. Pharmaceutics. 2021;13(2).
Puris E, Auriola S, Petralla S, Hartman R, Gynther M, de Lange ECM, et al. Altered protein expression of membrane transporters in isolated cerebral microvessels and brain cortex of a rat Alzheimer's disease model. Neurobiol Dis. 2022;169:105741.
Scalise M, Galluccio M, Console L, Pochini L, Indiveri C. The Human SLC7A5 (LAT1): The Intriguing Histidine/Large Neutral Amino Acid Transporter and Its Relevance to Human Health. Front Chem. 2018;6:243.
Huttunen J, Peltokangas S, Gynther M, Natunen T, Hiltunen M, Auriola S, et al. L-Type Amino Acid Transporter 1 (LAT1/Lat1)-Utilizing Prodrugs Can Improve the Delivery of Drugs into Neurons, Astrocytes and Microglia. Sci Rep. 2019;9(1):12860.
Sanchez-Contreras MY, Kouri N, Cook CN, Serie DJ, Heckman MG, Finch NA, et al. Replication of progressive supranuclear palsy genome-wide association study identifies SLCO1A2 and DUSP10 as new susceptibility loci. Mol Neurodegener. 2018;13(1):37.
Storck SE, Meister S, Nahrath J, Meissner JN, Schubert N, Di Spiezio A, et al. Endothelial LRP1 transports amyloid-beta(1-42) across the blood-brain barrier. J Clin Invest. 2016;126(1):123-36.
Rauch JN, Luna G, Guzman E, Audouard M, Challis C, Sibih YE, et al. LRP1 is a master regulator of tau uptake and spread. Nature. 2020;580(7803):381-5.
Kubanek J. Neuromodulation with transcranial focused ultrasound. Neurosurg Focus. 2018;44(2):E14.
Shin J, Kong C, Lee J, Choi BY, Sim J, Koh CS, et al. Focused ultrasound-induced blood-brain barrier opening improves adult hippocampal neurogenesis and cognitive function in a cholinergic degeneration dementia rat model. Alzheimers Res Ther. 2019;11(1):110.
Kim YG, Kim SE, Lee J, Hwang S, Yoo SS, Lee HW. Neuromodulation Using Transcranial Focused Ultrasound on the Bilateral Medial Prefrontal Cortex. J Clin Med. 2022;11(13).
Forster S, Thumser AE, Hood SR, Plant N. Characterization of rhodamine-123 as a tracer dye for use in in vitro drug transport assays. PLoS One. 2012;7(3):e33253.
Jouan E, Le Vee M, Mayati A, Denizot C, Parmentier Y, Fardel O. Evaluation of P-Glycoprotein Inhibitory Potential Using a Rhodamine 123 Accumulation Assay. Pharmaceutics. 2016;8(2).
Cioni C, Turlizzi E, Zanelli U, Oliveri G, Annunziata P. Expression of Tight Junction and Drug Efflux Transporter Proteins in an in vitro Model of Human Blood-Brain Barrier. Front Psychiatry. 2012;3:47.
Vogelgesang S, Warzok RW, Cascorbi I, Kunert-Keil C, Schroeder E, Kroemer HK, et al. The role of P-glycoprotein in cerebral amyloid angiopathy; implications for the early pathogenesis of Alzheimer's disease. Curr Alzheimer Res. 2004;1(2):121-5.
McCormick JW, Ammerman L, Chen G, Vogel PD, Wise JG. Transport of Alzheimer's associated amyloid-beta catalyzed by P-glycoprotein. PLoS One. 2021;16(4):e0250371.
Al Rihani SB, Darakjian LI, Deodhar M, Dow P, Turgeon J, Michaud V. Disease-Induced Modulation of Drug Transporters at the Blood-Brain Barrier Level. Int J Mol Sci. 2021;22(7).
Yamazaki Y, Kanekiyo T. Blood-Brain Barrier Dysfunction and the Pathogenesis of Alzheimer's Disease. Int J Mol Sci. 2017;18(9).
Mehta RI, Carpenter JS, Mehta RI, Haut MW, Ranjan M, Najib U, et al. Blood-Brain Barrier Opening with MRI-guided Focused Ultrasound Elicits Meningeal Venous Permeability in Humans with Early Alzheimer Disease. Radiology. 2021;298(3):654-62.
Rezai AR, Ranjan M, D'Haese PF, Haut MW, Carpenter J, Najib U, et al. Noninvasive hippocampal blood-brain barrier opening in Alzheimer's disease with focused ultrasound. Proc Natl Acad Sci U S A. 2020;117(17):9180-2.
Wang W, Bodles-Brakhop AM, Barger SW. A Role for P-Glycoprotein in Clearance of Alzheimer Amyloid beta -Peptide from the Brain. Curr Alzheimer Res. 2016;13(6):615-20.
Lee HJ, Ryu JM, Jung YH, Lee SJ, Kim JY, Lee SH, et al. High glucose upregulates BACE1-mediated Abeta production through ROS-dependent HIF-1alpha and LXRalpha/ABCA1-regulated lipid raft reorganization in SK-N-MC cells. Sci Rep. 2016;6:36746.
Badagnani I, Castro RA, Taylor TR, Brett CM, Huang CC, Stryke D, et al. Interaction of methotrexate with organic-anion transporting polypeptide 1A2 and its genetic variants. J Pharmacol Exp Ther. 2006;318(2):521-9.
Zhou F, Zheng J, Zhu L, Jodal A, Cui PH, Wong M, et al. Functional analysis of novel polymorphisms in the human SLCO1A2 gene that encodes the transporter OATP1A2. AAPS J. 2013;15(4):1099-108.
Leinenga G, Langton C, Nisbet R, Gotz J. Ultrasound treatment of neurological diseases--current and emerging applications. Nat Rev Neurol. 2016;12(3):161-74.
Szablewski L. Brain Glucose Transporters: Role in Pathogenesis and Potential Targets for the Treatment of Alzheimer's Disease. Int J Mol Sci. 2021;22(15).
Dvorak V, Wiedmer T, Ingles-Prieto A, Altermatt P, Batoulis H, Barenz F, et al. An Overview of Cell-Based Assay Platforms for the Solute Carrier Family of Transporters. Front Pharmacol. 2021;12:722889.
Profaci CP, Munji RN, Pulido RS, Daneman R. The blood-brain barrier in health and disease: Important unanswered questions. J Exp Med. 2020;217(4).

ChavesetalSupplementaryDoc.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Alzheimer’s disease brain endothelial-like cells reveal differential drug transporter expression and modulation by potentially therapeutic focused ultrasound

Status:

Version 1

Abstract

Figures

Background

Methods

Study design

Hipsc Expansion And Ibec Differentiation

Immunofluorescence For Hipsc And Ibec Characterization

Focused Ultrasound Treatment

Statistical Analysis

Results

Effects Of Fus Treatment On Pgp Activity In Control And Ad-ibecs

Effects Of Fus Treatment On Pgp-mediated Aβ Uptake In Control And Ad-ibecs

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1