The accumulation of amyloid-β peptide (Aβ) in the brain is a critical hallmark of Alzheimer's disease. This high cerebral Aβ concentration may be partly caused by impaired clearance of Aβ 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 Aβ across the BBB. We hypothesized that other ABC proteins, such as members of the G subfamily, are also involved in the BBB clearance of Aβ. We therefore investigated the roles of ABCG2 (BCRP) and ABCG4 in the efflux of [3H] Aβ1-40 from HEK293 cells stably transfected with human ABCG2 or mouse abcg4. We showed that ABCG2 and Abcg4 mediate the cellular efflux of [3H] Aβ1-40. In addition, probucol fully inhibited the efflux of [3H] Aβ1-40 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] Aβ1-40 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 Aβ at the mouse BBB. Perfusing the brains of Abcb1/Abcg2- and Abca1-deficient mice with [3H] Aβ1-40 plus probucol significantly increased the Clup of Aβ. This suggests that a probucol-sensitive transporter that is different from Abca1, Abcb1, and Abcg2 is involved in the brain efflux of Aβ. We suggest that this probucol-sensitive transporter is Abcg4. We conclude that Abcg4 acts in concert with Abcg2 to efflux Aβ from the brain across the BBB.

The blood-brain barrier restricts the passage of substances into the brain. Neuropeptides, such as enkephalins, cannot be delivered into the brain when given systemically because of this barrier. Therefore, there is a need to develop efficient transport systems to deliver these drugs to the brain. Recently, we have demonstrated that conjugation of doxorubicin or penicillin to peptide vectors significantly enhances their brain uptake. In this study, we have conjugated the enkephalin analog dalargin with two different peptide vectors, SynB1 and SynB3, to improve its brain delivery and its pharmacological effect. We show by in situ brain perfusion that vectorization markedly enhances the brain uptake of dalargin. We also show using the hot-plate model that this enhancement in brain uptake results in a significant improvement in the observed antinociceptive effect of dalargin. These results support the usefulness of peptide-mediated strategies for improving the availability and efficacy of central nervous system drugs.Brain delivery is one of the major challenges for the neuropharmaceutical industry since increasing number of hydrophilic therapeutic agents, such as anticancer drugs, antibiotics, and antiviral drugs are unable to cross the blood-brain barrier (BBB). The BBB represents a complex endothelial interface in vertebrates that separates the blood compartment from the extracellular fluid compartment of the brain parenchyma. The capillaries in the brain parenchyma possess a high electrical resistance due to tight junctions between the endothelial cells and also lack pores. Thus, the brain capillary endothelium behaves like a continuous lipid bilayer, and diffusion through this BBB layer is largely dependent on the lipid solubility of the drug. Because peptides are hydrophilic, biologically unstable, and large molecules, it is difficult for them to penetrate the BBB. Even though their brain uptake is not so high, some peptides and proteins are delivered into the brain by carrier-mediated transport, receptor-mediated transport, or adsorptive-mediated transport mechanisms. One of the problems associated with the inability of many peptides and proteins to accumulate in the brain in therapeutically meaningful amounts is the efflux transport systems. For example, it has been shown that the selective ␦-opioid receptors against [D-Pen 2 ,D-Pen 5 ]-enkephalin (DPDPE) has a poor BBB permeability that is explained in part by P-glycoprotein (P-gp)-mediated efflux, and DPDPE is also a substrate of the rat organic anion transporting polypeptide 2 (OATP2) and human OATP-A (Kakyo et al., 1999;Gao et al., 2000).To overcome the limited access of drugs to the brain, various strategies have been applied to direct central nervous system (CNS) drugs into the brain . Most of these methods are invasive, such as surgical implantation of an intraventricular catheter followed by drug infusion into the ventricular compartment, transient opening of the tight junctions by the intracarotid infusion of a hypertonic solution (Chamberl...

Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia–ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.

In the normal brain, immune cell trafficking and immune responses are strictly controlled and limited. This unique homeostatic equilibrium, also called brain immune quiescence, is crucial to maintaining proper brain functions and is altered in various pathological processes, from chronic immunopathological disorders to cognitive and psychiatric impairments. To date, the precise nature of factors regulating the brain/immune system interrelationship is poorly understood. In the present study, we demonstrate that one of these regulating factors is Connexin 43 (Cx43), a gap junction protein highly expressed by astrocytes at the blood-brain barrier (BBB) interface. We show that, by setting the activated state of cerebral endothelium, astroglial Cx43 controls immune recruitment as well as antigen presentation mechanisms in the mouse brain. Consequently, in the absence of astroglial Cx43, recruited immune cells elaborate a specific humoral autoimmune response against the von Willebrand factor A domain-containing protein 5a, an extracellular matrix protein of the brain. Altogether, our results demonstrate that Cx43 is a new astroglial factor promoting the immune quiescence of the brain.

Background:The rate of entry of cocaine into the brain is a critical factor that influences neuronal plasticity and the development of cocaine addiction. Until now, passive diffusion has been considered the unique mechanism known by which cocaine crosses the blood-brain barrier.Methods:We reassessed mechanisms of transport of cocaine at the blood-brain barrier using a human cerebral capillary endothelial cell line (hCMEC/D3) and in situ mouse carotid perfusion.Results:Both in vivo and in vitro cocaine transport studies demonstrated the coexistence of a carrier-mediated process with passive diffusion. At pharmacological exposure level, passive diffusion of cocaine accounted for only 22.5% of the total cocaine influx in mice and 5.9% in hCMEC/D3 cells, whereas the carrier-mediated influx rate was 3.4 times greater than its passive diffusion rate in vivo. The functional identification of this carrier-mediated transport demonstrated the involvement of a proton antiporter that shared the properties of the previously characterized clonidine and nicotine transporter. The functionnal characterization suggests that the solute carrier (SLC) transporters Oct (Slc22a1-3), Mate (Slc47a1) and Octn (Slc22a4-5) are not involved in the cocaine transport in vivo and in vitro. Diphenhydramine, heroin, tramadol, cocaethylene, and norcocaine all strongly inhibited cocaine transport, unlike benzoylecgonine. Trans-stimulation studies indicated that diphenhydramine, nicotine, 3,4-methylenedioxyamphetamine (ecstasy) and the cathinone compound 3,4-methylenedioxypyrovalerone (MDPV) were also substrates of the cocaine transporter.Conclusions:Cocaine transport at the BBB involves a proton-antiporter flux that is quantitatively much more important than its passive diffusion. The molecular identification and characterization of this transporter will provide new tools to understand its role in addictive mechanisms.

This study investigated the effects of docosahexaenoic acid (DHA)-rich phospholipid supplementation on behavior, electroretinogram and phospholipid fatty acid (PUFA) composition in selected brain regions and retina in old mice. Two groups of mice were fed a semisynthetic balanced diet or a diet deficient in alpha-linolenic acid. At the age of 8 months, half of each diet group was supplemented with DHA. In the open field, no differences in motor or exploratory activities were observed between the four diet groups. In the light/dark test of anxiety, the time spent in the light compartment was significantly higher in both supplemented groups than in control and deficient groups. Learning performance in the Morris water maze was significantly impaired in deficient old mice, but was completely restored by the phospholipid supplementation. The electroretinogram showed a significant alteration of a- and b-wave amplitudes in control compared to deficient mice. Phospholipid supplementation induced a significant increase of b-wave amplitude in both control and deficient groups and restored normal fatty acid composition in brain regions and retina in deficient mice. DHA-rich phospholipids may improve learning ability, visual function and reverse biochemical modifications in old mice fed an n-3 polyunsaturated fatty acid-deficient diet; they also may improve visual function in old mice fed a balanced diet.

Abstract. Nicotine, the main tobacco alkaloid leading to smoking dependence, rapidly crosses the bloodbrain barrier (BBB) to become concentrated in the brain. Recently, it has been shown that nicotine interacts with some organic cation transporters (OCT), but their influence at the BBB has not yet been assessed in vivo. In this study, we characterized the transport of nicotine at the mouse luminal BBB by in situ brain perfusion. Its influx was saturable and followed the Michaelis-Menten kinetics (K m 02.60 mM, V max 037.60 nmol/s/g at pH 7.40). At its usual micromolar concentrations in the plasma, most (79%) of the net transport of nicotine at the BBB was carrier-mediated, while passive diffusion accounted for 21%. Studies on knockout mice showed that the OCT Oct1-3, P-gp, and Bcrp did not alter [3 H]-nicotine transport at the BBB. Neither did inhibiting the transporters Mate1, Octn, or Pmat. The in vivo manipulation of intracellular and/or extracellular pH, the chemical inhibition profile, and the transstimulation experiments demonstrated that the nicotine transporter at the BBB shared the properties of the clonidine/proton antiporter. The molecular features of this proton-coupled antiporter have not yet been identified, but it also transports diphenhydramine and tramadol and helps nicotine cross the BBB at a faster rate and to a greater extent. The pharmacological inhibition of this nicotine/proton antiporter could represent a new strategy to reduce nicotine uptake by the brain and thus help curb addiction to smoking.

Uptake1 and uptake2 transporters are involved in the extracellular clearance of biogenic amine neurotransmitters at synaptic clefts. We looked for them at the blood-brain barrier (BBB) and bloodretina barriers (BRB), where they could be involved in regulating the neurotransmitter concentration and modulate/terminate receptor-mediated effects within the neurovascular unit (NVU). Uptake2 (Oct1-3/ Slc22a1-3, Pmat/Slc29a4) and Mate1/Slc47a1 transporters are also involved in the transport of xenobiotics. We used in situ carotid perfusion of prototypic substrates like-serotonin, and [ 3 H]-dopamine, changes in ionic composition and genetic deletion of Oct1-3 carriers to detect uptake1 and uptake2 at the BBB and BRB. We showed that uptake1 and uptake2 are involved in the transport of [ 3 H]-dopamine and [ 3 H]-MPP + at the blood luminal BRB, but not at the BBB. These functional studies, together with quantitative RT-PCR and confocal imaging, suggest that the mouse BBB lacks uptake1 (Net/Slc6a2, Dat/Slc6a3, Sert/Slc6a4), uptake2, and Mate1 on both the luminal and abluminal sides. However, we found evidence for functional Net and Oct1 transporters at the luminal BRB. These heterogeneous transport properties of the brain and retina NVUs suggest that the BBB helps protect the brain against biogenic amine neurotransmitters in the plasma while the BRB has more of a metabolic/endocrine role.