McCaffrey, Gwen PhD; Davis, Thomas P. PhD
DYNAMIC NATURE OF THE BLOOD-BRAIN BARRIER
The blood-brain barrier (BBB) is the critical boundary between the central nervous system (CNS) and the periphery. It is both friend and foe to the clinician. In safeguarding the CNS from exposure to potentially harmful substances in the systemic circulation, the BBB simultaneously presents a serious obstacle to CNS drug delivery. Anatomically, the BBB is a vast network of ∼650 km of microcapillaries, each of which has a lumen diameter of ∼6 mm and is encircled by a single, nonfenestrated endothelial cell whose luminal (blood-facing) and abluminal (brain-facing) membranes are separated by ∼300 to 500 nm of cytoplasm.1–3 Substances attempting to pass from the systemic circulation to the brain must take the paracellular route between or the transcellular route through the microvascular endothelial cells. Paracellular diffusion of water-soluble substances and small ions is severely restricted by tight junctions (TJs) that connect apposing endothelial cell membranes to physically obliterate the interendothelial cleft. Blood-borne substances attempting to pass through the luminal membrane of microvascular endothelial cells are actively expelled back into the capillary lumen by embedded efflux transporters, or acted upon by a variety of metabolizing enzymes. The combined efforts of passive obstruction (TJs), active drug efflux (embedded transporters), and biochemical transformation (metabolism) create an obstacle to drug delivery that prevents approximately 98% of small molecule drugs and essentially 100% of large molecule drugs (e.g., monoclonal antibodies, antisense drugs) from entering the brain under normal conditions.4–7
The BBB is not a static anatomical boundary, but a dynamic interface capable of rapid response to stressors including hypoxia, inflammation, trauma, and pain.3,4,8–11 Therapeutic targeting of the BBB is emerging as a critically relevant clinical goal3,8,9,11–13 because BBB dysfunction exacerbates (and in selected instances, perhaps initiates14) numerous diseases and pathologies including stroke,11,15–17 Alzheimer disease,18–23 acute liver failure,24 multiple sclerosis,25,26 meningitis,27,28 HIV,29–31 diabetes,32–34 depressive and psychotic disorders,35 cerebral malaria,36 Parkinson disease,22,26 traumatic10,23,37–40 and surgical41 brain injury, peripheral nerve injury,42 brain cancer,43–45 epilepsy,46–49 and peripheral inflammatory pain.3,8,50 Loss of BBB integrity (i.e., leak) exposes the brain to potentially harmful concentrations of substances in the peripheral circulation (e.g., ions, amino acids, neurotransmitters, proteins, and other macromolecules) that may disrupt brain homeostasis and adversely affect neuronal signaling. Inappropriate paracellular passage of therapeutic pharmaceuticals, nutraceuticals, or xenobiotics into the brain following TJ disruption may result in significant drug adverse effects and/or adverse drug-drug interactions. Alternatively, BBB impairment may involve pathologically increased drug efflux across the microvascular luminal membrane that results in reduced drug uptake into the brain and diminished drug efficacy (Fig. 1).
PROTEIN TRAFFICKING AS A THERAPEUTIC TARGET TO MODULATE BBB FUNCTION
Blood-brain barrier integrity and function are critically influenced by what is now referred to as the “extended neurovascular unit”51 that incorporates not only microvascular endothelial cells and adjacent pericytes, astrocytes, and neurons, but also neighboring smooth muscle cells and microglia in the brain and blood cells in the capillary lumen such as polymorphonuclear cells, lymphocytes, and monocytes.3,4,12,14,52 Given the multiplicity of cell types, intracellular and extracellular signaling pathways, and interacting proteins, lipids, and carbohydrates involved in the formation, maintenance, and disruption of the different barrier functions performed by the BBB, there are a multitude of approaches for therapeutic manipulation of the BBB in both health and disease to optimize CNS drug delivery. At the molecular level, an established approach to influence the function of a particular protein important to BBB biochemistry (e.g., efflux transporter, TJ component) is a direct modulation of its activity and/or gene transcription. An alternative approach for enhancing CNS drug delivery that is under study in our laboratory is the targeting of protein trafficking whereby altering the location of a protein is used as the means of modulating its activity. The unique advantage to therapeutic subcellular misdirection (or redirection) of a protein is that its physiological impact can be reversibly modified, despite pathology-induced changes in gene transcription.53 Discussed below are 2 examples from our laboratory, involving the drug efflux transporter P-glycoprotein and the TJ transmembrane protein occludin, that demonstrate the potential of therapeutic modulation of pathology-induced changes in BBB protein trafficking to optimize CNS drug delivery in the presence of stressors (e.g., peripheral inflammatory pain, hypoxia).
ALTERED P-GLYCOPROTEIN TRAFFICKING PROMOTES INCREASED DRUG EFFLUX AT THE BBB
P-glycoprotein (ABCB1/MDR1, EC 18.104.22.168) is the preeminent molecular challenge to CNS drug delivery at the BBB.54–56 Strategically enriched at the luminal membrane of cerebral microvascular endothelial cells,57–59 P-glycoprotein uses energy from ATP hydrolysis to expel an impressive variety of structurally divergent drugs back into the microcapillary lumen against steep concentration gradients. P-glycoprotein substrates range in mass from ∼300 to 4000 Da and include analgesics, anticancer and immunosuppressive agents, psychotropics, antibiotics, antiallergenics, antiepileptics, β-blockers, steroid hormones, and HIV-1 protease inhibitors.54,55,60–64 Although intense research effort has focused on the development of P-glycoprotein inhibitors, clinical trials incorporating direct inhibition of P-glycoprotein have largely proved unsuccessful in improving therapeutic efficacy.65–70 High doses of inhibitor appear to be required, unfortunately giving rise to systemic toxicity. Moreover, complete inhibition of P-glycoprotein could be life-threatening because of the lack of protection against potentially dangerous blood-borne substances. Currently, research effort is focused on identifying therapeutic targets within multiple signaling pathways that promote disease-related changes in P-glycoprotein activity.45–47,54,55,62,71–73
In our laboratory, we discovered that the onset of peripheral inflammatory pain (experimentally induced by injection of λ-carrageenan in the rat hind paw) is followed within 3 hours by an increase in P-glycoprotein–associated efflux of morphine at the BBB. The important consequence of this was a corresponding decrease in morphine efficacy in vivo due to a reduction in morphine uptake into the brain.74 These data demonstrated that inflammatory pain itself hinders the ability of clinically relevant pain drugs such as morphine to gain entrance into the brain. Inflammation caused by tissue injury contributes to the severity of postoperative pain,75 and therefore our finding of increased morphine efflux by P-glycoprotein at the BBB may explain in part the reported difficulties with achieving postoperative opioid analgesia.76,77 To identify novel strategies to overcome significant decreases in CNS analgesic drug delivery that occur following the onset of peripheral inflammatory pain, we investigated the hypothesis that the rapid increase in P-glycoprotein efflux function was due to a dynamic redistribution of P-glycoprotein within the microvascular endothelial cell wherein P-glycoprotein stored within a putative reservoir was released from storage and trafficked to the luminal plasma membrane. Evolutionary selection of P-glycoprotein to be a critical “gatekeeper” at the BBB can be inferred by the fact that microvessels at the BBB contain the highest levels of P-glycoprotein within the body78 and that in vivo dosing studies using P-glycoprotein substrates show that brain uptake is substantially increased in P-glycoprotein knockout animals.79,80 Given that diet, environmental exposure or external stressors can quickly raise the concentration of potentially harmful substances in the systemic circulation, we surmised that as P-glycoprotein evolved to perform so significant a barrier role at the BBB, a mechanism must also have evolved to ensure the timely trafficking of sufficient amounts of P-glycoprotein to the microvascular luminal membrane to meet the current threat.
Biosynthetic trafficking of P-glycoprotein in vivo at the BBB has not been studied. As an N-linked glycosylated protein, P-glycoprotein can be assumed within microvascular endothelial cells to follow the classic anterograde biosynthetic pathway, with cotranslational insertion into the lumen of the endoplasmic reticulum being followed by glycosylation, folding, additional posttranslational modification in the Golgi, and, finally, transport to the plasma membrane.81,82 Protein transport from one subcellular location to another directly results from specific protein-protein interactions that are governed by unique motifs encoded within a protein’s primary sequence. P-glycoprotein has a binding motif in its N-terminus for caveolin-1,83 a key trafficking protein capable of forming both caveolar and noncaveolar oligomeric scaffolds.84 Caveolin-1 colocalizes with P-glycoprotein in caveolae isolated from rat brain capillaries85 and at the luminal endothelial membrane and the border of the luminal/abluminal compartments in human brain capillaries.59,86 Studies using rat brain endothelial cells in vitro have demonstrated that the physical interaction between P-glycoprotein with caveolin-1 is enhanced by tyrosine-14-phosphorylation of caveolin-1 and that the binding of P-glycoprotein to caveolin-1 negatively regulates P-glycoprotein function.87,88
To investigate the constitutive and inflammation/pain-induced trafficking of P-glycoprotein within cerebral microvascular endothelial cells in vivo, we used the λ-carrageenan model of inflammatory pain (i.e., hyperalgesia), combined with confocal microscopy and subcellular fractionation of isolated cerebral microvessels.89 Quantitative microscopic global colocalization examination of intact cerebral microvessels revealed that under normal conditions there was a significant amount of colocalization between P-glycoprotein and caveolin-1. Subcellular fractionation of isolated cerebral microvessel homogenate revealed that P-glycoprotein trafficking is highly regulated, with the bulk of the transporter apparently being targeted to and sequestered within high-molecular-weight (>250 kDa) “storage” structures enriched in caveolin-1 and maintained by disulfide bonds. Biochemical analysis of isolated membrane domainsenriched with P-glycoprotein–containing high-molecular-weight structures revealed that these apparent reservoirs of P-glycoprotein quickly disassembled in the presence of a hydrophilic reducing agent, indicating that the cysteine residues forming the structural disulfide bonds were readily accessible to the external milieu. Incorporation of P-glycoprotein within densely packed, high-molecular-weight complexes would protect against both limited proteolysis and proteasomal or lysosomal degradation. Moreover, the sensitivity of the P-glycoprotein–containing high-molecular-weight structures to reduction by a hydrophilic reducing agent suggested that this manner of “storing” P-glycoprotein involved its sequestration within a structure that could readily be completely dismantled to release a large amount of monomeric, biologically active P-glycoprotein at one time. Peripheral inflammatory pain reduced the colocalization of P-glycoprotein with caveolin-1 within cerebral microvessels by approximately half within 3 hours of onset and promoted a dramatic redistribution of P-glycoprotein and caveolin-1 between endothelial cell subcellular compartments. Disassembly of high-molecular-weight structures containing P-glycoprotein coincided with an increase in drug-stimulated P-glycoprotein–dependent ATPase activity associated with plasma membrane domains identified to be at the luminal surface of cerebral microvessels. These data are the first observation that peripheral inflammatory pain leads to altered trafficking of P-glycoprotein that is responsible for controlling analgesic drug delivery to the brain. Future biochemical analysis of isolated P-glycoprotein–containing high-molecular-weight “storage” complexes will allow identification of potential therapeutic targets for maintaining the integrity of high-molecular-weight complexes storing P-glycoprotein. Thus, optimizing CNS drug delivery to the brain during pathological states such as peripheral inflammatory pain could be achieved by preventing release of P-glycoprotein from subcellular storage compartments.
INHIBITION OF OCCLUDIN TRAFFICKING AWAY FROM TJS RESTORES BBB INTEGRITY
The transmembrane protein occludin is critical for barrier function at TJs between microvascular endothelial cells at the BBB,3,90 and trafficking of occludin away from TJs is a sensitive, early, and reliable sign of TJ opening and BBB dysfunction.91 The ability to rapidly seal BBB breaches (i.e., leaks) at TJs that occur during oxidative stress (e.g., stroke) would preclude the development of life-threatening cerebral edema and the inappropriate CNS delivery of neurotoxic blood-borne substances.9,11,16,51 In our laboratory, we discovered that peripheral inflammatory pain induced in 3 different experimental models (formalin, carrageenan, and complete Freund’s adjuvant) promoted BBB dysfunction characterized by increased paracellular permeability to vascular markers such as sucrose.92–96 Peripheral inflammatory pain was also found to promote an increase in paracellular permeability of the opioid analgesic codeine.97 Codeine analgesia is centrally mediated, which requires it to accumulate within the brain, and it does so by passive paracellular diffusion.98,99 An uncontrolled increase in brain uptake of codeine during peripheral inflammatory pain due to a pathological increase in paracellular permeability may result in significant CNS adverse effects associated with opioids such as respiratory depression, addiction, and tolerance. Alterations in BBB TJ barrier function have now been reported in experimental pain models incorporating chronic spinal nerve ligation100 and peripheral nerve injury,42 highlighting the need to develop a means of targeting increases in paracellular permeability to optimize CNS drug delivery of clinically relevant pain drugs such as codeine that passage paracellularly across the BBB.
Occludin has an M-shaped topology, fashioned by 4 transmembrane domains, 2 extracellular loops, and cytoplasmic N- and C-termini, which facilitates its performance of both structural and signaling roles at the TJ.90,101–103 Through its extracellular loops, occludin physically extends into the interendothelial space to interact with homologous segments of occludin molecules on adjacent microvascular endothelial cells to help fuse apposing cell membranes to form a tight seal that restricts paracellular diffusion.104,105 Occludin is capable of self-association,106–108 and occludin oligomerization is facilitated by the presence in its primary sequence of a motif of ∼200 amino acids that has statistical similarity to the myelin and lymphocyte (MAL) and related proteins for vesicle trafficking and membrane link (MARVEL).109,110 Through its C-terminus, occludin interacts with TJ accessory proteins such as the zonula occludens proteins (ZO-1, ZO-2, and ZO-3) that anchor multiprotein TJ complexes to the underlying actin cytoskeleton.111,112 At different sites within the N- and C-termini and the intracellular loop that are exposed to the cytoplasm, occludin interacts with a variety of signaling, regulatory, and vesicle trafficking proteins including numerous kinases and phosphatases,101,113 growth factor receptors,114,115 caveolin,116 rab13,117 the ubiquitin-protein ligase itch,118 and proteins containing a ubiquitin interacting motif (Epsin, epidermal growth factor receptor pathway substrate 15 and hepatocyte growth factor–regulated tyrosine kinase).119
To understand, on a molecular level, how occludin performs both structural and signaling roles at BBB TJs, it was first necessary to develop a means of isolating native TJ complexes containing oligomeric assemblies of occludin. Early freeze-fracture replica electron microscopy studies revealed TJs to be continuous, anastomosing, intramembranous particle strands of ∼10-nm thickness, giving rise to the belief that tightly packed oligomeric assemblies of integral membrane proteins are an essential architectural feature of the TJ.120–122 To avoid disruption of occludin oligomeric assemblies during their isolation, we incorporated the use of a novel detergent-free, density gradient method123 to fractionate cerebral microvessels. This made it possible, for the first time, to isolate occludin oligomeric complexes within the context of their normal lipid-enriched plasma membrane raft environment.124 Biochemical analysis of isolated occludin oligomeric assemblies derived from naive rats revealed the importance of disulfide bonds in maintaining the structural integrity of these large-molecular-weight structures and provided an explanation for the evolutionary conservation of cysteine residues in the occludin molecule found within hydrophobic transmembrane regions and hydrophilic regions such as the second extracellular loop and the cytoplasmic C-terminus.125 Occludin oligomeric assemblies were found to contain 2 sets of disulfide bonds, distinguished by the ease with which they could be reduced by either a hydrophilic or a hydrophobic reducing agent. Although high-molecular-weight occludin complexes were sensitive to a hydrophilic reducing agent that disrupts easily accessible disulfide bonds, complete disruption of occludin oligomeric assemblies could be achieved only by treatment with a stronger hydrophobic reducing agent capable of penetrating the hydrophobic, transmembrane regions of occludin multiprotein complexes to reach buried disulfide bonds. Our data generated the hypothesis that disassembly of oligomeric occludin complexes within “TJ-associated” plasma membrane lipid rafts could be initiated by selective reduction of readily accessible disulfide bonds involving cysteine residues within extracellular loops of occludin molecules on apposing cell membranes, or perhaps within the cytoplasmic C-termini of adjacent occludin molecules within the same cell. Conformational changes promoted by relaxation of structural restrictions invoked by disulfide bonds could then lead to altered protein-lipid interactions, a remodeling of the lipid raft environment, and a progressive dismantling of the oligomeric complex into component lower-molecular-weight occludin isoforms.
Biochemical analysis of occludin oligomeric complexes, isolated from cerebral microvessels from experimental animals subjected to either peripheral inflammatory pain or global hypoxia/reoxygenation, provided additional data to refine our model of protein isoform interaction within occludin oligomeric assemblies.96,126,127 Occludin oligomeric assemblies at BBB TJs were revealed to have an inner “structural core” of covalently bonded subunits that was associated through noncovalent, hydrophobic interactions with a variety of monomeric and dimeric subunits. Disruption of a disulfide bond(s) between occludin molecules on different sides of the paracellular cleft caused by an eternal stressor conceivably could lead to conformational changes in selected subunits within the inner core that resulted in a physical breach in the transmembrane protein diffusion barrier. The change in conformation of specific occludin isoforms within the center of the oligomeric complex would expectedly lead to a change in conformation of selected non–covalently bound subunits, rendering the latter more accessible to signaling and regulatory molecules and/or more readily disassociated from the parent oligomeric occludin structure.
To provide evidence that limited disulfide reduction due to oxidative stress was a precipitating event leading to the trafficking of occludin away from “TJ-associated” occludin oligomeric assemblies, we investigated if TJ disruption and altered occludin trafficking could be modulated by an antioxidant. Using the noninvasive in vivo rat model of global ischemia, we found that the membrane-permeable, free radical scavenger TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) preserved the integrity of disulfide bonds within occludin oligomeric assemblies and prevented an increase in paracellular permeability to sucrose.11,127 TEMPOL also prevented carrageenan-induced peripheral inflammatory pain from inducing TJ disruption and altered trafficking of occludin and an associated increase in paracellular permeability to codeine.8,95 These data demonstrated that use of antioxidants such as TEMPOL to protect the “Achilles heel of occludin oligomeric assemblies”96 (i.e., sensitivity to disulfide-bond reduction during oxidative stress103) is a viable means of therapeutically manipulating occludin trafficking in vivo to optimize CNS drug delivery by preventing the dismantling of multiprotein TJ complexes and the associated changes in paracellular permeability at the BBB.
Protein trafficking and the development of pathological conditions and disease are closely linked.128–130 Although the development of pharmaceutical “relocators and mislocators”53 for the therapeutic modulation of protein trafficking is in its infancy, recent clinical trials incorporating the use of the lipid-based compound perifosine131 to prevent antiapoptotic kinase (AKT) activation by inhibiting its trafficking to the plasma membrane highlight the promise that clinical targeting of protein trafficking can offer to the treatment of colorectal cancer and multiple myeloma132 and possibly also neuroblastoma.133 Protein-protein interactions, and the changes in protein trafficking that they promote, are increasingly being recognized as an important means of regulating CNS proteins such as opioid receptors,134–137 AMPA receptors,138 nicotinic acetylcholine receptors,139 glutamate receptors,140 tyrosine kinase receptors,141 cannabinoid CB1 receptors,142 and other G protein coupled receptors143,144 and vesicular neurotransmitter transporters.145 Methodology to identify motifs within primary sequences of proteins that regulate protein-protein interactions is rapidly advancing in sophistication and scope.146–153 In addition, research efforts to develop small molecules and peptides for clinical use as manipulators of the protein-protein interactions that drive changes in protein trafficking are greatly intensifying.53,147,153–161 By designing the protein-protein interaction inhibitor to preferentially inhibit the interaction of 2 specific proteins, as opposed to the interaction of one protein with a variety of substrates, then specificity of protein trafficking changes can be achieved within minimal adverse effects.154
Therapeutic targeting of protein-protein interactions that lead to changes in key BBB efflux transporter or TJ protein trafficking and localization is a relatively unexplored means of optimizing CNS drug delivery. In the example of P-glycoprotein, temporary reduction of its activity at the plasma membrane can be achieved by inhibiting its release from a storage reservoir, thus providing a short-term window for drug delivery at the BBB. Trafficking modulation can also be longer term, as in the case of using a pharmacological reactive oxygen species scavenger such as TEMPOL to inhibit the oxidative stress–induced disruption of disulfide bonds within occludin oligomeric assemblies that promotes the trafficking of occludin isoforms away from the TJ and the development of increased paracellular permeability. As detailed understanding of the trafficking mechanisms involved in trafficking of key BBB efflux transporter and TJ proteins increases, so will the identification of novel targets for therapeutic modulation of BBB integrity and function (Fig. 2).
Using the parameters of effectiveness, safety, and cost of application to rank a drug delivery strategy,162 enhancement of CNS drug delivery at the BBB through therapeutic manipulation of efflux transporter trafficking (e.g., P-glycoprotein) ranks favorably as it has the potential to provide a noninvasive and low-cost means of increasing the entry of a wide variety of drugs into the brain under conditions of disease or stress without compromising basal BBB protection. This stands in contrast to the high cost of treatment and patient safety concerns associated with artificial opening of the BBB (e.g., with mannitol), which may lead to uncontrolled CNS entry of potentially harmful substances present in the systemic circulation along with the target drug.18,163,164 Central nervous system drug delivery strategies using chemical delivery systems (e.g., liposomes, nanocarriers, conjugates) or endocytosis and transcytosis (adsorptive and receptor mediated) are similar in concept to therapeutic manipulation of P-glycoprotein trafficking in that they provide a means of circumventing P-glycoprotein drug efflux, but these approaches have potentially high formulation costs and significant patient safety concerns. However, with further development, these technologies have the potential to provide a targeted approach for the delivery of specific drugs to the CNS.161,163,165–171 In contrast, therapeutic manipulation of P-glycoprotein trafficking represents a relatively inexpensive and nonspecific approach for enhancing CNS drug delivery of a multitude of drugs and drug combinations.
The authors thank Dr. William D. Staatz for his helpful advice on this article and his expert assistance with illustration preparation.
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