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Administration of synthetic or purified peptides directly into the brain ventricles is a method commonly used by neuroscientists for exploring physiological and behavioral functions of gene products. i.v. administration is controlled by the blood-brain barrier, which limits its effectiveness, and current approaches for acute or chronic intracerebroventricular delivery have significant technical drawbacks resulting from both the chemical properties of the delivered substance and the experimental procedures. Here we describe a genetic approach for the delivery of secreted peptides or proteins into the cerebrospinal fluid (CSF). Using a choroid plexus-specific promoter, we established a lentiviral-based system, which offers inducible and reversible delivery of a gene product into the CSF. The functionality of this system was demonstrated by using the overexpression of the two established neuropeptides, corticotropin-releasing factor and gonadotropin-releasing hormone, modulating anxiety-like behavior and estrus cycle, respectively. We show that this choroid plexus-specific lentiviral-based system is a reliable, effective, and adaptable research tool for intracerebroventricular delivery. blood--brain barrier | choroid plexus | Lentiviruses | secreted peptides | cerebrospinal fluid doi/10.1073/pnas.0907059107

Drug abuse is a worldwide health concern in which addiction involves activation of the dopaminergic signaling pathway in the brain. Here, we introduce a nanotechnology approach that utilizes gold nanorod-DARPP-32 siRNA complexes (nanoplexes) that target this dopaminergic signaling pathway in the brain. The shift in the localized longitudinal plasmon resonance peak of gold nanorods (GNRs) was used to show their interaction with siRNA. Plasmonic enhanced dark field imaging was used to visualize the uptake of these nanoplexes in dopaminergic neurons in vitro. Gene silencing of the nanoplexes in these cells was evidenced by the reduction in the expression of key proteins (DARPP-32, ERK, and PP-1) belonging to this pathway, with no observed cytotoxicity. Moreover, these nanoplexes were shown to transmigrate across an in vitro model of the blood-brain barrier (BBB). Therefore, these nanoplexes appear to be suited for brain-specific delivery of appropriate siRNA for therapy of drug addiction and other brain diseases. DARPP-32 | dark field imaging | surface plasmon resonance | nanoplexes | blood-brain barrier

Treatment of many neuronal degenerative disorders will require delivery of a therapeutic protein to neurons or glial cells across the whole CNS. The presence of the blood--brain barrier hampers the delivery of these proteins from the blood, thus necessitating a new method for delivery. Receptors on the blood--brain barrier bind ligands to facilitate their transport to the CNS; therefore, we hypothesized that by targeting these receptors, we may be able to deliver proteins to the CNS for therapy. Here, we report the use of the lentivirus vector system to deliver the lysosomal enzyme glucocerebrosidase and a secreted form of GFP to the neurons and astrocytes in the CNS. We fused the low-density lipoprotein receptor-binding domain of the apolipoprotein B to the targeted protein. This approach proved to be feasible for delivery of the protein and could possibly be used as a general method for delivery of therapeutic proteins to the CNS. fusion protein | lentiviral vectors | low-density lipoprotein receptor

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the CNS that is used to model certain parameters of multiple sclerosis. To establish the relative contributions of T cell reactivity, the loss of blood-brain barrier (BBB) integrity, CNS inflammation, and lesion formation toward the pathogenesis of EAE, we assessed the incidence of EAE and these parameters in mice lacking NF-[kappa]B, TNF-[alpha], IFN-[alpha][beta] receptors, IFN-[gamma] receptors, and inducible nitric oxide synthase. Although increased myelin oligodendrocyte glycoprotein-specific T cell reactivity was generally associated with a more rapid onset or increased disease severity, the loss of BBB integrity and cell accumulation in spinal cord tissues was invariably associated with the development of neurological disease signs. Histological and real-time RT-PCR analyses revealed differences in the nature of immune/inflammatory cell accumulation in the spinal cord tissues of the different mouse strains. On the other hand, disease severity during the acute phase of EAE directly correlated with the extent of BBB permeability. Thus, the loss of BBB integrity seems to be a requisite event in the development of EAE and can occur in the absence of important inflammatory mediators. gene knockout mice | multiple sclerosis

Mucopolysaccharidosis type VII is a lysosomal storage disorder resulting from inherited deficiency of [beta]-glucuronidase (GUS). Mucopolysaccharidosis type VII is characterized by glycosaminoglycan storage in most tissues, including brain. In these disorders, enzyme delivery across the blood-brain barrier (BBB) is the main obstacle to correction of lysosomal storage in the CNS. Prior studies suggested mouse brain is accessible to GUS in the first 2 weeks of life but not later. To explore a possible role for the mannose 6-phosphate/insulin-like growth factor II receptor in GUS transport across the BBB in neonatal mice, we compared brain uptake of phosphorylated GUS (P-GUS) and nonphosphorylated GUS (NP-GUS) in newborn and adult mice. [sup.131]I-P-GUS was transported across the BBB after i.v. injection in 2-day-old mice. The brain influx rate ([K.sub.in]) of [sup.131]I-P-GUS in 2-day-old mice was 0.21 [micro]l/g*min and decreased with age. By 7 weeks of age, transport of [sup.131]I-P-GUS was not significant. Capillary depletion revealed that 62% of the [sup.131]I-P-GUS in brain was in brain parenchyma in 2-day-old mice. In addition, uptake of [sup.131]I-P-GUS into brain was significantly reduced by coinjection of unlabeled P-GUS or M6P in a dose-dependent manner, in contrast, the [K.sub.in] of [sup.131]I-NP-GUS (0.04 [micro]l/g*min) was significantly lower than [sup.131]I-P-GUS in 2-day-old mice. Transcardiac brain perfusion confirmed that neither [sup.131]I-P-GUS nor [sup.131]I-NP-GUS crossed the BBB in adult mice. These results indicate that [sup.131]I-P-GUS transport into brain parenchyma in early postnatal life is mediated by the mannose 6-phosphate/insulin-like growth factor II receptor. This receptor-mediated transport is not observed in adult mice. beta-glucuronidase | mannose 6-phosphate/insulin-like growth factor II receptor | central nervous system | lysosomal storage disease | phosphorylated [beta]-glucuronidase

The blood-brain barrier and a blood - cerebrospinal-fluid (CSF) barrier function together to isolate the brain from circulating drugs, toxins, and xenobiotics. The blood - CSF drug-permeability barrier is localized to the epithelium of the choroid plexus (CP). However, the molecular mechanisms regulating drug permeability across the CP epithelium are defined poorly. Herein, we describe a drug-permeability barrier in human and rodent CP mediated by epithelial-specific expression of the MDR1 (multidrug resistance) P glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP). Noninvasive single-photon.emission computed tomography with 99mTc-sestamibi, a membrane-permeant radiopharmaceutical whose transport is mediated by both Pgp and MRP, shows a large blood-to-CSF concentration gradient across intact CP epithelium in humans in vivo. In rats, pharmacokinetic analysis with 99mTc-sestamibi determined the concentration gradient to be greater than 100-fold. In membrane fractions of isolated native CP from rat, mouse, and human, the 170-kDa Pgp and 190-kDa MRP are identified readily. Furthermore, the murine proteins are absent in CP isolated from their respective mdr1a/1b(-/-) and mrp(-/-) gene knockout littermates. As determined by immunohistochemical and drug-transport analysis of native CP and polarized epithelial cell cultures derived from neonatal rat CP, Pgp localizes subapically, conferring an apical-to-basal transepithelial permeation barrier to radiolabeled drugs. Conversely, MRP localizes basolaterally, conferring an opposing basal-to-apical drug-permeation barrier. Together, these transporters may coordinate secretion and reabsorption of natural product substrates and therapeutic drugs, including chemotherapeuric agents, antipsychotics, and HIV protease inhibitors, into and out of the central nervous system.

In vivo antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood-brain barrier penetration rate. In vitro, isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing [Na.sup.+] ions from the incubation medium decreases the carrier affinity for deltorphins (-25%), but does not affect the [V.sub.max] value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective [Delta]-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for [Delta]- or [Mu]-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that [Delta]-, and [Kappa]-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with L-glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.

A soluble form of Alzheimer disease amyloid [Beta]-protein (sA[Beta]) is transported in the blood and cerebrospinal fluid mainly complexed with apolipoprotein J (apoJ). Using a well-characterized in situ perfused guinea pig brain model, we recently obtained preliminary evidence that apoJ facilitates transport of sA[[Beta].sub.1-40]-apoJ complexes across the blood-brain barrier and the blood-cerebrospinal fluid barrier, but the mechanisms remain poorly understood. In the present study, we examined the transport process in greater detail and investigated the possible role of glycoprotein 330 (gp330)/megalin, a receptor for multiple ligands, including apoJ. High-affinity transport systems with a Km of 0.2 and 0.5 nM were demonstrated for apoJ at the blood-brain barrier and the choroid epithelium in vivo, suggesting a specific receptor-mediated mechanism. The sA[[Beta].sub.1-40]-apoJ complex shared the same transport mechanism and exhibited 2.4- to 10.2-fold higher affinity than apoJ itself. Binding to microvessels, transport into brain parenchyma, and choroidal uptake of both apoJ and sA[[Beta].sub.1-40]-apoJ complexes were markedly inhibited (74-99%) in the presence of a monoclonal antibody to gp330/megalin and were virtually abolished by perfusion with the receptor-associated protein, which blocks binding of all known ligands to gp330. Western blot analysis of cerebral microvessels with the monoclonal antibody to gp330 revealed a protein with a mass identical to that in extracts of kidney membranes enriched with gp330/megalin, but in much lower concentration. The findings suggest that gp330/megalin mediates cellular uptake and transport of apoJ and sA[[Beta].sub.1-40]-apoJ complex at the cerebral vascular endothelium and choroid epithelium.

The transport of solutes between blood and brain is regulated by a specific barrier. Capillary endothelial cells of brain are known to mediate barrier function and facilitate transport. Here we report that specific cells surrounding arterioles, known as Mato's fluorescent granular perithelial (FGP) cells or perivascular microglial cells, contribute to the barrier function. Immunohistochemical and in situ hybridization studies indicate that, in normal brain cortex, type I and type II macrophage scavenger receptors are expressed only in FGP/perivascular microglial cells, and surface markers of macrophage lineage are also detected on them. These cells mediate the uptake of macromolecules, including modified low density lipoprotein, horseradish peroxidase, and ferritin injected either into the blood or into the cerebral ventricles. Accumulation of scavenged materials with aging or after the administration of a high-fat diet results in the formation of honeycomb-like foam cells and the narrowing of the lumen of arterioles in the brain cortex. These results indicate involvement of FGP/perivascular microglial cells in the barrier and scavenger functions in the central nervous system.

Intraperitoneally injected analogues of glycopeptide enkephalin penetrate the blood-brain barrier and bind to the delta- and mu-opioid receptors in mouse brain. Glycosylation reduces the biophilic nature of the peptides and enhances the transport functions of lipophilic endothelial layer. The analogues induce analgesia in the mice.

An in vivo strategy adopts shake-flask methods or HPLC to measure the partition equilibrium of a drug, in transition state between an aqueous and an immiscible phase, facilitates the functional characterization of drugs to permeate the blood-brain barrier. The drug's amphiphilicity determines its surface activity which is quantified by its Gibbs adsorption isotherms. A calibration graph for critical micelle concentration as a function of micelle levels required for the drug's surface activity can be used to predict the central nervous system permeability of a drug.

Nicotine was methylated with charged methyl iodide to produce two quaternized and permanently charged cationic derivatives. The effects of quaternization on brain uptake was investigated using high-pressure liquid chromatography and nuclear magnetic resonance spectroscopy. The results showed that N-methyl quaternization of nicotine markedly reduced its penetration in the blood-brain barrier. This makes N-methylnicotines undesireable addictive products.

Previous investigations have demonstrated that among the various isoforms of carbonic anhydrase (CA), CA II is intrinsically associated with nervous tissue. However, immunohistochemistry and immunoelectron microscopy on sections of rat and mouse brain demonstrated that CA IV is also associated with nervous tissue. In contrast to the oligodendrocyte-specific distribution of CA II, CA IV is associated with the luminal surface of capillary endothelial cells. This suggests that CA IV plays a functional role in the blood-brain barrier.

Brain cells depend on the availability of glucose for energy metabolism. Glucose transport to the brain can be assessed by utilizing plasma levels to measure brain glucose concentrations. Direct measurement by rapid-freezing in vitro is only possible in animals and not in humans. However, NMR spectroscopy has proven its ability to assess the concentration of several metabolites in living tissue. This method allows direct measurement of brain glucose concentrations and the possibility of examining glucose transport control in human brain in vivo appears promising.

Antisense radiopharmaceuticals could be used to image gene expression in the brain in vivo, should these polar molecules be made transportable through the blood-brain barrier. The present studies describe an antisense imaging agent comprised of an iodinated peptide nucleic acid (PNA) conjugated to a monoclonal antibody to the rat transferrin receptor by using avidin-biotin technology. The PNA was a 16-mer antisense to the sequence around the methionine initiation codon of the luciferase mRNA. C6 rat glioma cells were permanently transfected with a luciferase expression plasmid, and C6 experimental brain tumors were developed in adult rats. The expression of the luciferase transgene in the tumors in vivo was confirmed by measurement of luciferase enzyme activity in the tumor extract. The [1251]PNA conjugate was injected intravenously in anesthetized animals with brain tumors and killed 2 h later for frozen sectioning of brain and film auto-radiography. No image of the luciferase gene expression was obtained after the administration of either the unconjugated antiluciferase PNA or a PNA conjugate that was antisense to the mRNA of a viral transcript. In contrast, tumors were imaged in all rats administered the [1251]PNA that was antisense to the luciferase sequence and was conjugated to the targeting antibody. In conclusion, these studies demonstrate gene expression in the brain in vivo can be imaged with antisense radiopharmaceuticals that are conjugated to a brain drug-targeting system. blood-brain barrier | transferrin receptor | monoclonal antibody | peptide nucleic acid

Erythropoietin (EPO), recognized for its central role in erythropoiesis, also mediates neuroprotection when the recombinant form (r-Hu-EPO) is directly injected into ischemic rodent brain. We observed abundant expression of the EPO receptor at brain capillaries, which could provide a route for circulating EPO to enter the brain. In confirmation of this hypothesis, systemic administration of r-Hu-EPO before or up to 6 h after focal brain ischemia reduced injury by [approximately equals] 50-75%. R-Hu-EPO also ameliorates the extent of concussive brain injury, the immune damage in experimental autoimmune encephalomyelitis, and the toxicity of kainate. Given r-Hu-EPO's excellent safety profile, clinical trials evaluating systemically administered r-Hu-EPO as a general neuroprotective treatment are warranted.

Gene therapy of the brain is hindered by the presence of the blood-brain barrier (BBB), which prevents the brain uptake of bloodborne gene formulations. Exogenous genes have been expressed in the brain after invasive routes of administration, such as craniotomy or intracarotid arterial infusion of noxious agents causing BBB disruption. The present studies describe the expression of an exogenous gene in brain after noninvasive i.v. administration of a 6- to 7-kb expression plasmid encoding either luciferase or [Beta]-galactosidase packaged in the interior of neutral pegylated immunoliposomes. The latter are conjugated with the OX26 mAb to the rat transferrin receptor, which enables targeting of the plasmid DNA to the brain via the endogenous BBB transferrin receptor. Unlike cationic liposomes, this neutral liposome formulation is stable in blood and does not result in selective entrapment in the lung. Luciferase gene expression in the brain peaks at 48 h after a single i.v. administration of 10 [micro]g of plasmid DNA per adult rat, a dose that is 30- to 100-fold lower than that used for gene expression in rodents with cationic liposomes, [Beta]-Galactosidase histochemistry demonstrated gene expression throughout the central nervous system, including neurons, choroid plexus epithelium, and the brain microvasculature. In conclusion, widespread gene expression in the brain can be achieved by using a formulation that does not employ viruses or cationic liposomes, but instead uses endogenous receptor-mediated transport pathways at the BBB. blood-brain barrier | transferrin receptor | monoclonal antibody | gene therapy