How ATP suppresses the fibrillation of amyloid peptides: analysis of the free-energy contributions.

Recent experiments have revealed that adenosine triphosphate (ATP) suppresses the fibrillation of amyloid peptides - a process closely linked to neurodegenerative diseases such as Alzheimer's and Parkinson's. Apart from the adsorption of ATP onto amyloid peptides, the molecular understanding is still limited, leaving the underlying mechanism for the fibrillation suppression by ATP largely unclear, especially in regards to the molecular energetics. Here we provide an explanation at the molecular scale by quantifying the free energies using all-atom molecular dynamics simulations. We found that the changes of the free energies due to the addition of ATP lead to a significant equilibrium shift towards monomeric peptides in agreement with experiments. Despite ATP being a highly charged species, the decomposition of the free energies reveals that the van der Waals interactions with the peptide are decisive in determining the relative stabilization of the monomeric state. While the phosphate moiety exhibits strong electrostatic interactions, the compensation by the water solvent results in a minor, overall Coulomb contribution. Our quantitative analysis of the free energies identifies which intermolecular interactions are responsible for the suppression of the amyloid fibril formation by ATP and offers a promising method to analyze the roles of similarly complex cosolvents in aggregation processes.

An innovative strategy to identify new targets for delivering antibodies to the brain has led to the exploration of the integrin family.

BACKGROUND: Increasing brain exposure of biotherapeutics is key to success in central nervous system disease drug discovery. Accessing the brain parenchyma is especially difficult for large polar molecules such as biotherapeutics and antibodies because of the blood-brain barrier. We investigated a new immunization strategy to identify novel receptors mediating transcytosis across the blood-brain barrier. METHOD: We immunized mice with primary non-human primate brain microvascular endothelial cells to obtain antibodies. These antibodies were screened for their capacity to bind and to be internalized by primary non-human primate brain microvascular endothelial cells and Human Cerebral Microvascular Endothelial Cell clone D3. They were further evaluated for their transcytosis capabilities in three in vitro blood-brain barrier models. In parallel, their targets were identified by two different methods and their pattern of binding to human tissue was investigated using immunohistochemistry. RESULTS: 12 antibodies with unique sequence and internalization capacities were selected amongst more than six hundred. Aside from one antibody targeting Activated Leukocyte Cell Adhesion Molecule and one targeting Striatin3, most of the other antibodies recognized ß1 integrin and its heterodimers. The antibody with the best transcytosis capabilities in all blood-brain barrier in vitro models and with the best binding capacity was an anti-αnß1 integrin. In comparison, commercial anti-integrin antibodies performed poorly in transcytosis assays, emphasizing the originality of the antibodies derived here. Immunohistochemistry studies showed specific vascular staining on human and non-human primate tissues. CONCLUSIONS: This transcytotic behavior has not previously been reported for anti-integrin antibodies. Further studies should be undertaken to validate this new mechanism in vivo and to evaluate its potential in brain delivery.

Tetravalent Bispecific Tandem Antibodies Improve Brain Exposure and Efficacy in an Amyloid Transgenic Mouse Model.

Most antibodies display very low brain exposure due to the blood-brain barrier (BBB) preventing their entry into brain parenchyma. Transferrin receptor (TfR) has been used previously to ferry antibodies to the brain by using different formats of bispecific constructs. Tetravalent bispecific tandem immunoglobulin Gs (IgGs) (TBTIs) containing two paratopes for both TfR and protofibrillar forms of amyloid-beta (Aß) peptide were constructed and shown to display higher brain penetration than the parent anti-Aß antibody. Additional structure-based mutations on the TfR paratopes further increased brain exposure, with maximal enhancement up to 13-fold in wild-type mice and an additional 4-5-fold in transgenic (Tg) mice harboring amyloid plaques, the main target of our amyloid antibody. Parenchymal target engagement of extracellular amyloid plaques was demonstrated using in vivo and ex vivo fluorescence imaging as well as histological methods. The best candidates were selected for a chronic study in an amyloid precursor protein (APP) Tg mouse model showing efficacy at reducing brain amyloid load at a lower dose than the corresponding monospecific antibody. TBTIs represent a promising format for enhancing IgG brain penetration using a symmetrical construct and keeping bivalency of the payload antibody.

Non-Human Primate Blood-Brain Barrier and In Vitro Brain Endothelium: From Transcriptome to the Establishment of a New Model.

The non-human primate (NHP)-brain endothelium constitutes an essential alternative to human in the prediction of molecule trafficking across the blood-brain barrier (BBB). This study presents a comparison between the NHP transcriptome of freshly isolated brain microcapillaries and in vitro-selected brain endothelial cells (BECs), focusing on important BBB features, namely tight junctions, receptors mediating transcytosis (RMT), ABC and SLC transporters, given its relevance as an alternative model for the molecule trafficking prediction across the BBB and identification of new brain-specific transport mechanisms. In vitro BECs conserved most of the BBB key elements for barrier integrity and control of molecular trafficking. The function of RMT via the transferrin receptor (TFRC) was characterized in this NHP-BBB model, where both human transferrin and anti-hTFRC antibody showed increased apical-to-basolateral passage in comparison to control molecules. In parallel, eventual BBB-related regional differences were investigated in seven-day in vitro-selected BECs from five brain structures: brainstem, cerebellum, cortex, hippocampus, and striatum. Our analysis retrieved few differences in the brain endothelium across brain regions, suggesting a rather homogeneous BBB function across the brain parenchyma. The presently established NHP-derived BBB model closely mimics the physiological BBB, thus representing a ready-to-use tool for assessment of the penetration of biotherapeutics into the human CNS.

Age-Dependent Regulation of the Blood-Brain Barrier Influx/Efflux Equilibrium of Amyloid-ß Peptide in a Mouse Model of Alzheimer's Disease (3xTg-AD).

The involvement of transporters located at the blood-brain barrier (BBB) has been suggested in the control of cerebral Aß levels, and thereby in Alzheimer's disease (AD). However, little is known about the regulation of these transporters at the BBB in animal models of AD. In this study, we investigated the BBB expression of Aß influx (Rage) and efflux (Abcb1-Abcg2-Abcg4-Lrp-1) transporters and cholesterol transporter (Abca1) in 3-18-month-old 3xTg-AD and control mice. The age-dependent effect of BBB transporters regulation on the brain uptake clearance (Clup) of [3H]cholesterol and [3H]Aß1 - 40 was then evaluated in these mice, using the in situ brain perfusion technique. Our data suggest that transgenes expression led to the BBB increase in Aß influx receptor (Rage) and decrease in efflux receptor (Lrp-1). Our data also indicate that mice have mechanisms counteracting this increased net influx. Indeed, Abcg4 and Abca1 are up regulated in 3- and 3/6-month-old 3xTg-AD mice, respectively. Our data show that the balance between the BBB influx and efflux of Aß is maintained in 3 and 6-month-old 3xTg-AD mice, suggesting that Abcg4 and Abca1 control the efflux of Aß through the BBB by a direct (Abcg4) or indirect (Abca1) mechanism. At 18 months, the BBB Aß efflux is significantly increased in 3xTg-AD mice compared to controls. This could result from the significant up-regulation of both Abcg2 and Abcb1 in 3xTg-AD mice compared to control mice. Thus, age-dependent regulation of several Aß and cholesterol transporters at the BBB could ultimately limit the brain accumulation of Aß.

Altered cerebral vascular volumes and solute transport at the blood-brain barriers of two transgenic mouse models of Alzheimer's disease.

We evaluated the integrity and function of the blood-brain barrier in 3xTg-AD mice aged 3-18 months and in APP/PS1 mice aged 8-months to determine the impacts of changes in amyloid and tau proteins on the brain vascular changes. The vascular volume (Vvasc) was sub-normal in 3xTg-AD mice aged from 6 to 18 months, but not in the APP/PS1 mice. The uptakes of [(3)H]-diazepam by the brains of 3xTg-AD, APP/PS1 and their age-matched control mice were similar at all the times studied, suggesting that the simple diffusion of small solutes is unchanged in transgenic animals. The uptake of d-glucose by the brains of 18-month old 3xTg-AD mice, but not by those of 8-month old APP/PS1 mice, was reduced compared to their age-matched controls. Accordingly, the amount of Glut-1 protein was 1.4 times lower in the brain capillaries of 18 month-old 3xTg-AD mice than in those of age-matched control mice. We conclude that the brain vascular volume is reduced early in 3xTg-AD mice, 6 months before the appearance of pathological lesions, and that this reduction persists until they are at least 18 months old. The absence of alterations in the BBB of APP/PS1 mice suggests that hyperphosphorylated tau proteins contribute to the vascular changes that occur in AD.

Oatp1a4 and an L-thyroxine-sensitive transporter mediate the mouse blood-brain barrier transport of amyloid-ß peptide.

The influx of amyloid-ß peptide (Aß) across the blood-brain barrier is partly mediated by the receptor for advanced glycation end products (RAGE). But other transporters, like Oatp (organic anion transporter polypeptide, SLC21) transporters, could also be involved. We used in situ brain perfusion to show that rosuvastatin and taurocholate, two established Oatp1a4 substrates, decreased (5-fold) the Clup of [3H]Aß while L-thyroxine increased it (5.5-fold). We demonstrated an interaction between Aß and Oatp1a4 by co-immunoprecipitation and western blotting experiments, supporting the hypothesis that the rosuvastatin- and taurocholate-sensitive transporter was Oatp1a4. In conclusion, our results suggest that, in mice, the brain uptake of Aß is partly mediated by Oatp1a4 and that L-thyroxine may play a crucial role in the inhibition of brain Aß clearance.

ABCG2- and ABCG4-mediated efflux of amyloid-ß peptide 1-40 at the mouse blood-brain barrier.

The accumulation of amyloid-ß peptide (Aß) in the brain is a critical hallmark of Alzheimer's disease. This high cerebral Aß concentration may be partly caused by impaired clearance of Aß across the blood-brain barrier (BBB). The low-density lipoprotein receptor-related protein-1 (LRP-1) and the ATP-binding cassette (ABC) protein ABCB1 (P-glycoprotein) are involved in the efflux of Aß across the BBB. We hypothesized that other ABC proteins, such as members of the G subfamily, are also involved in the BBB clearance of Aß. We therefore investigated the roles of ABCG2 (BCRP) and ABCG4 in the efflux of [3H] Aß1-40 from HEK293 cells stably transfected with human ABCG2 or mouse abcg4. We showed that ABCG2 and Abcg4 mediate the cellular efflux of [3H] Aß1-40. In addition, probucol fully inhibited the efflux of [3H] Aß1-40 from HEK293-abcg4 cells. Using the in situ brain perfusion technique, we showed that GF120918 (dual inhibitor of Abcb1 and Abcg2) strongly enhanced the uptake (Clup, µl/g/s) of [3H] Aß1-40 by the brains of Abcb1-deficient mice, but not by the brains of Abcb1/Abcg2-deficient mice, suggesting that Abcg2 is involved in the transport of Aß at the mouse BBB. Perfusing the brains of Abcb1/Abcg2- and Abca1-deficient mice with [3H] Aß1-40 plus probucol significantly increased the Clup of Aß. This suggests that a probucol-sensitive transporter that is different from Abca1, Abcb1, and Abcg2 is involved in the brain efflux of Aß. We suggest that this probucol-sensitive transporter is Abcg4. We conclude that Abcg4 acts in concert with Abcg2 to efflux Aß from the brain across the BBB.

Direct evidence of abca1-mediated efflux of cholesterol at the mouse blood-brain barrier.

We investigated the expression and function of Abca1 in wild-type C57BL/6, abca1(+/+), and abca1(-/-) mice brain capillaries forming the blood-brain barrier (BBB). We first demonstrated by quantitative RT-PCR and Western immunoblot that Abca1 was expressed and enriched in the wild-type mouse brain capillaries. In abca1(-/-) mice, we reported that the lack of Abca1 resulted in an 1.6-fold increase of the Abcg4 expression level compared to abca1(+/+) mice. Next, using the in situ brain perfusion technique, we showed that the [(3)H]cholesterol brain uptake clearance (Cl(up), µl/s/g brain), was significantly increased (107%) in abca1(-/-) mice compared to abca1(+/+) mice, meaning that the deficiency of Abca1 conducted to a significant decrease of the cholesterol efflux at the BBB level. In addition, the co-perfusion of probucol (Abca1 inhibitor) with [(3)H]cholesterol resulted in an increase of [(3)H]cholesterol Cl(up) (115%) in abca1(+/+) but not in abca1(-/-) mice, meaning that probucol inhibited selectively the efflux function of Abca1. In conclusion, our results demonstrated that Abca1 was expressed in the mouse brain capillaries and that Abca1 functions as an efflux transporter through the mouse BBB.

Reduction of the cerebrovascular volume in a transgenic mouse model of Alzheimer's disease.

Combined evidence from neuroimaging and neuropathological studies shows that signs of vascular pathology and brain hypoperfusion develop early in Alzheimer's disease (AD). To investigate the functional implication of these abnormalities, we have studied the cerebrovascular volume and selected markers of blood-brain barrier (BBB) integrity in 11-month-old 3 x Tg-AD mice, using the in situ brain perfusion technique. The cerebrovascular volume of distribution of two vascular space markers, [3H]-inulin and [14C]-sucrose, was significantly lower (-26% and -27%, respectively; p < 0.01) in the brain of 3 x Tg-AD mice compared to non-transgenic littermates. The vascular volume reduction was significant in the hippocampus (p < 0.01), but not in the frontal cortex and cerebellum. However, the brain transport coefficient (Clup) of [14C]-D-glucose (1 microM) and [3H]-diazepam was similar between 3xTg-AD mice and controls, suggesting no difference in the functional integrity of the BBB. We also report a 32% increase (p < 0.001) in the thickness of basement membranes surrounding cortical microvessels along with a 20% increase (p < 0.05) of brain collagen content in 3xTg-AD mice compared to controls. The present data indicate that the cerebrovascular space is reduced in a mouse model of Abeta and tau accumulation, an observation consistent with the presence of cerebrovascular pathology in AD.