Modifications of physical and functional integrity of the blood-brain barrier in an inducible mouse model of neurodegeneration.

The inducible p25 overexpression mouse model recapitulate many hallmark features of Alzheimer's disase including progressive neuronal loss, elevated Aß, tau pathology, cognitive dysfunction, and impaired synaptic plasticity. We chose p25 mice to evaluate the physical and functional integrity of the blood-brain barrier (BBB) in a context of Tau pathology (pTau) and severe neurodegeneration, at an early (3 weeks ON) and a late (6 weeks ON) stage of the pathology. Using in situ brain perfusion and confocal imaging, we found that the brain vascular surface area and the physical integrity of the BBB were unaltered in p25 mice. However, there was a significant 14% decrease in cerebrovascular volume in 6 weeks ON mice, possibly explained by a significant 27% increase of collagen IV in the basement membrane of brain capillaries. The function of the BBB transporters GLUT1 and LAT1 was evaluated by measuring brain uptake of d-glucose and phenylalanine, respectively. In 6 weeks ON p25 mice, d-glucose brain uptake was significantly reduced by about 17% compared with WT, without any change in the levels of GLUT1 protein or mRNA in brain capillaries. The brain uptake of phenylalanine was not significantly reduced in p25 mice compared with WT. Lack of BBB integrity, impaired BBB d-glucose transport have been observed in several mouse models of AD. In contrast, reduced cerebrovascular volume and an increased basement membrane thickness may be more specifically associated with pTau in mouse models of neurodegeneration.

Effect of citrulline on muscle protein turnover in an in vitro model of muscle catabolism.

OBJECTIVE: Muscle net catabolism, as seen after severe trauma or sepsis or in postoperative situations, is mediated by hormones (e.g., cortisol) and proinflammatory cytokines (e.g., tumor necrosis factor alpha [TNF-α]). Specific amino acids may be able to limit this muscle mass loss. Citrulline (CIT) stimulates muscle protein synthesis in various situations, but little data exist on hypercatabolic situations and the effects on protein breakdown are unknown. Our aim was to assess the effect of CIT on protein turnover in an in vitro model of muscle hypercatabolism. METHODS: Myotubes derived from C2C12 myoblasts were treated with 150 nM dexamethasone (DEX), 10 ng/mL TNF-α, or 0.006% ethanol (as control [CON]) for 24 h. Subsequently, myotubes were incubated with or without 5 mM CIT for 6 h. Muscle protein synthesis rate was evaluated by the surface sensing of translation method and by l-[3,5-3H]tyrosine (Tyr) incorporation. The muscle protein breakdown rate was evaluated from Tyr release into culture medium. CIT action was analyzed by non-parametric Kruskal-Wallis and Mann-Whitney tests. RESULTS: CIT treatment significantly increased protein synthesis rates compared with the DEX or TNF-α group (surface sensing of translation method; DEX + CIT versus DEX; P = 0.03 and TNF-α+CIT versus TNF-α; P = 0.05) and significantly decreased protein breakdown rate in the CON and DEX groups (CON + CIT versus CON; P = 0.05 and DEX + CIT versus DEX; P = 0.05). CONCLUSIONS: CIT treatment regulated muscle protein turnover in an in vitro model of muscle net catabolism. Exploring the underlying mechanisms would also be of interest.

High brain distribution of a new central nervous system drug candidate despite its P-glycoprotein-mediated efflux at the mouse blood-brain barrier.

Efficacy of drugs aimed at treating central nervous system (CNS) disorders rely partly on their ability to cross the cerebral endothelium, also called the blood-brain barrier (BBB), which constitutes the main interface modulating exchanges of compounds between the brain and blood. In this work, we used both, conventional pharmacokinetics (PK) approach and in situ brain perfusion technique to study the blood and brain PK of PKRinh, an inhibitor of the double-stranded RNA-dependent protein kinase (PKR) activation, in mice. PKRinh showed a supra dose-proportional blood exposure that was not observed in the brain, and a brain to blood AUC ratio of unbound drug smaller than 1 at all tested doses. These data suggested the implication of an active efflux at the BBB. Using in situ brain perfusion technique, we showed that PKRinh has a very high brain uptake clearance which saturates with increasing concentrations. Fitting the data to a Michaelis-Menten equation revealed that PKRinh transport through the BBB is composed of a passive unsaturable flux and an active saturable protein-mediated efflux with a km of ≅ 3 µM. We were able to show that the ATP-binding cassette (ABC) transporter P-gp (Abcb1), but not Bcrp (Abcg2), was involved in the brain to blood efflux of PKRinh. At the circulating PKRinh concentrations of this study, the P-gp was not saturated, in accordance with the linear brain PKRinh PK. Finally, PKRinh had high brain uptake clearance (14 µl/g/s) despite it is a good P-gp substrate (P-gp Efflux ratio ≅ 3.6), and reached similar values than the cerebral blood flow reference, diazepam, in P-gp saturation conditions. With its very unique brain transport properties, PKRinh improves our knowledge about P-gp-mediated efflux across the BBB for the development of new CNS directed drugs.

Expression and function of Abcg4 in the mouse blood-brain barrier: role in restricting the brain entry of amyloid-ß peptide.

ABCG4 is an ATP-binding cassette transmembrane protein which has been shown, in vitro, to participate in the cellular efflux of desmosterol and amyloid-ß peptide (Aß). ABCG4 is highly expressed in the brain, but its localization and function at the blood-brain barrier (BBB) level remain unknown. We demonstrate by qRT-PCR and confocal imaging that mouse Abcg4 is expressed in the brain capillary endothelial cells. Modelling studies of the Abcg4 dimer suggested that desmosterol showed thermodynamically favorable binding at the putative sterol-binding site, and this was greater than for cholesterol. Additionally, unbiased docking also showed Aß binding at this site. Using a novel Abcg4-deficient mouse model, we show that Abcg4 was able to export Aß and desmosterol at the BBB level and these processes could be inhibited by probucol and L-thyroxine. Our assay also showed that desmosterol antagonized the export of Aß, presumably as both bind at the sterol-binding site on Abcg4. We show for the first time that Abcg4 may function in vivo to export Aß at the BBB, in a process that can be antagonized by its putative natural ligand, desmosterol (and possibly cholesterol).

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.