GFRalpha3 is expressed predominantly in nociceptive sensory neurons.

Activation of the RET receptor tyrosine kinase by glial-derived neurotrophic factor family members is dependent on a family of coreceptors, GFRalpha1-4. GFRalpha3 preferentially binds the newest member of the glial-derived neurotrophic factor family of ligands, artemin. The major site of GFRalpha3 expression is in the dorsal root ganglion; however, the class of sensory neurons that expresses GFRalpha3 has not been reported previously. Using immunohistochemical methods, we show that the majority of dorsal root ganglion cells that express GFRalpha3 also express vanilloid receptor type 1, peripherin, RET, trkA and calcitonin gene-related peptide. In addition, a significant subpopulation of GFRalpha3-expressing cells also binds the lectin IB4. We demonstrate that GFRalpha3 artemin neurons are immunopositive for markers expected of nociceptors and include a subset of neurons distinct from the GDNF-responsive population. Our results indicate artemin may exert selective effects on pain sensation.

Developmental regulation of GDNF response and receptor expression in the enteric nervous system.

The development of the enteric nervous system is dependent upon the actions of glial cell line-derived neurotrophic factor (GDNF) on neural crest-derived precursor cells in the embryonic gut. GDNF treatment of cultured enteric precursor cells leads to an increase in the number of neurons that develop and/or survive. Here we demonstrate that, although GDNF promoted an increase in neuron number at all embryonic ages examined, there was a developmental shift from a mitogenic to a trophic response by the developing enteric neurons. The timing of this shift corresponded to developmental changes in gut expression of GFR alpha-1, a co-receptor in the GDNF-Ret signaling complex. GFR alpha-1 was broadly expressed in the gut at early developmental stages, at which times soluble GFR alpha-1 was released into the medium by cultured gut cells. At later times, GFR alpha-1 became restricted to neural crest-derived cells. GFR alpha-1 could participate in GDNF signaling when expressed in cis on the surface of enteric precursor cells, or as a soluble protein. The GDNF-mediated response was greater when cell surface, compared with soluble, GFR alpha-1 was present, with the maximal response seen the presence of both cis and trans forms of GFR alpha-1. In addition to contributing to GDNF signaling, cell-surface GFR alpha-1 modulated the specificity of interactions between GDNF and soluble GFR alphas. These experiments demonstrate that complex, developmentally regulated, signaling interactions contribute to the GDNF-dependent development of enteric neurons.

Glial cell line-derived neurotrophic factor-dependent RET activation can be mediated by two different cell-surface accessory proteins.

Glial cell line-derived neurotrophic factor (GDNF)-dependent activation of the tyrosine kinase receptor RET is necessary for kidney and enteric neuron development, and mutations in RET are associated with human diseases. Activation of RET by GDNF has been shown to require an accessory component, GDNFR-alpha (RETL1). We report the isolation and characterization of rat and human cDNAs for a novel cell-surface associated accessory protein, RETL2, that shares 49% identity with RETL1. Both RETL1 and RETL2 can mediate GDNF dependent phosphorylation of RET, but they exhibit different patterns of expression in fetal and adult tissues. The most striking differences in expression observed were in the adult central and peripheral nervous systems. In addition, the mechanisms by which the two accessory proteins facilitate the activation of RET by GDNF are quite distinct. In vitro binding experiments with soluble forms of RET, RETL1 and RETL2 demonstrate that while RETL1 binds GDNF tightly to form a membrane-associated complex which can then interact with RET, RETL2 only forms a high affinity complex with GDNF in the presence of RET. This strong RET dependence of the binding of RETL2 to GDNF was confirmed by FACS analysis on RETL1 and RETL2 expressing cells. Together with the recent discovery of a GDNF related protein, neurturin, these data raise the possibility that RETL1 and RETL2 have distinctive roles during development and in the nervous system of the adult. RETL1 and RETL2 represent new candidate susceptibility genes and/or modifier loci for RET-associated diseases.

A competitive chemiluminescent enzyme-linked immunosorbent assay for the determination of RMP-7 in human blood.

RMP-7, a bradykinin agonist, is a synthetic nonapeptide designed to enhance the delivery of therapeutics to the central nervous system. A sensitive, competitive chemiluminescent enzyme-linked immunosorbent assay (ELISA) for quantifying RMP-7 in human blood samples has been developed. Rabbit antibodies against RMP-7 were produced using the conjugate of RMP-7 to keyhole limpet hemocyanin through glutaraldehyde. Biotinylated RMP-7, conjugated via N-hydroxysuccinimide ester, was used as the tracer. A premixed solution of biotinylated alkaline phosphatase and avidin was used to quantify the tracer, with a dioxetane-based compound as the chemiluminescent substrate. The method involves treating blood samples with organic solvents to precipitate proteins, evaporating the supernatants to dryness, reconstituting residues in PBS and assaying the buffer solutions with the ELISA. The assay, using 1.0 ml of whole blood, has precision and accuracy within +/- 20% over the concentration range 25-800 pg ml-1. There are no significant endogenous interferences. The assay has been successfully used to support clinical trials of RMP-7.

Characterization, receptor mapping and blood-brain barrier transcytosis of antibodies to the human transferrin receptor.

Monoclonal antibodies to the human transferrin receptor were screened for binding to capillary vessels in human, monkey, rabbit and rat brain tissue. Two antibodies were selected that bind both human and monkey but not rabbit or rat microvessels. With recombinant fragments of the human receptor, both antibodies were shown to bind to a region of the extracellular portion of the receptor that is relatively variable among species. Binding, which was characterized by using purified receptor and K562 cells, was not reduced by excess transferrin, indicating that the antibodies bind the receptor at a site different from that of transferrin. When the antibodies were radiolabeled and injected i.v. into cynomolgous monkeys, they distributed selectively to brain but not to other organs or tissues. The antibodies were found almost exclusively in the brain parenchyma, rather than the capillaries, indicating that they had transcytosed the blood-brain barrier. These results show that antibodies to the human transferrin receptor cross the blood-brain barrier and may be useful for noninvasive delivery of therapeutic proteins to the central nervous system.

Enhanced uptake of rsCD4 across the rodent and primate blood-brain barrier after conjugation to anti-transferrin receptor antibodies.

The delivery to the brain of nonlipophilic therapeutic compounds, especially proteins, is severely hindered by the presence of the blood-brain barrier, which is formed by the tightly apposed brain capillary endothelial cells. However, brain endothelial cells do possess specific receptor-mediated transport mechanisms so that substances required by the brain can cross the blood-brain barrier. By use of monoclonal antibodies that bind to the transferrin receptor present on the luminal surface of brain capillary endothelial cells, we have taken advantage of the transport system responsible for the delivery of iron to the brain to deliver recombinant human soluble CD4 (rsCD4), a potential anti-HIV therapeutic, across the blood-brain barrier. Anti-transferrin receptor antibody-rsCD4 conjugates were synthesized with a disulfide linkage and characterized in vitro. Experiments that use immunohistochemistry to localize these conjugates after intravenous administration into the tail vein of rats have shown that both the carrier antibody and the protein "passenger" accumulate in brain capillaries. The carrier-mediated delivery of radiolabeled protein across the blood-brain barrier in vivo was also examined in both rodents and primates. With use of the technique of capillary depletion in rats, the amount of rsCD4 in the capillary fraction of the brain, which reaches a maximal value within 1 hr postinjection, was shown to decrease with time, whereas the amount in the brain parenchyma increased, which suggests that the protein was delivered across the blood-brain barrier. In primates rsCD4 levels in the brain were increased 5-fold when the protein was administrated intravenously in the form of an anti-transferrin receptor antibody-rsCD4 conjugate.

Effects of transferrin receptor antibody-NGF conjugate on young and aged septal transplants in oculo.

The purpose of this study was to investigate the effects of nerve growth factor (NGF) conjugated to a monoclonal transferrin receptor antibody (OX-26) on septal transplants in oculo. Three different doses of OX-26-NGF conjugate (0.3, 3, and 50 micrograms/injection) were injected into the tail vein of young adult hosts 2, 4, and 6 weeks following intraocular transplantation of fetal forebrain tissue containing septal nuclei. Intravenous injections of OX-26 alone, NGF alone, and saline served as controls. An increase in intraocular tissue growth, as well as an increase in the intensity of immunoreactivity for p75 receptors and acetylcholinesterase, was observed following peripheral OX-26-NGF administration at the two highest doses tested. In addition, aged host rats with 16-month-old intraocular septal grafts were injected intravenously with OX-26 or OX-26-NGF (10 micrograms NGF/injection) every 2 weeks until the transplants were 24 months old. The intensity of choline acetyltransferase-like (ChAT) staining appeared to be greater and the cell bodies were larger with more processes in aged transplants in hosts treated with the OX-26-NGF conjugate than in aged OX-26-treated subjects. The present results suggest that peripheral OX-26-NGF can deliver biologically active NGF across the blood-brain barrier and have dose-dependent positive effects on both aged and developing cholinergic neurons in septal transplants.

Intravenous administration of a transferrin receptor antibody-nerve growth factor conjugate prevents the degeneration of cholinergic striatal neurons in a model of Huntington disease.

Intrastriatal injections of quinolinic acid induce a pattern of neuronal degeneration similar to that seen in Huntington disease. In the present study, nerve growth factor (NGF) crossed the blood-brain barrier in a dose-dependent fashion following intravenous infusion when conjugated to an antibody directed against the transferrin receptor (OX-26). Intravenous injections of the OX-26-NGF conjugate selectively prevented the loss of striatal choline acetyltransferase-immunoreactive neurons which normally occurs following quinolinic acid administration relative to control rats receiving vehicle or a nonconjugated mixture of OX-26 and NGF. These data demonstrate that a neurotrophic factor-antibody conjugate can prevent the degeneration of central NGF-responsive neurons following systemic administration.

NGF and anti-transferrin receptor antibody conjugate: short and long-term effects on survival of cholinergic neurons in intraocular septal transplants.

We describe a new molecular carrier system that allows for the transport of nerve growth factor (NGF) across the blood-brain barrier (BBB), as assessed by trophic effects on intraocular forebrain transplants that contain central cholinergic neurons. The carrier system involves monoclonal antibodies (OX-26) directed against the transferrin receptor, to which NGF molecules are covalently linked. Transferrin receptors are highly concentrated on brain blood vessels and participate in the transport of iron across the BBB. Host rats with septal transplants were divided into four groups, which received OX-26-NGF, OX-26, NGF or saline intravenously at 2, 4, 6 and 8 weeks after grafting. Half of the animals were killed directly after the final injection, whereas the other half were allowed to survive for an additional 5 months. Control experiments revealed that blood vessels in mature brain grafts in oculo contained large amounts of transferrin receptors. Covalent binding of NGF to the OX-26 antibodies did not impede OX-26 binding to CNS transferrin receptors, nor did conjugation affect the bioactivity of NGF. A time-dependent increase in host brain NGF levels was found after injection of OX-26-NGF into the tail vein. Host serum contained some NGF antibodies in the short-term OX-26-NGF group that had disappeared in the long-term group; host adrenals showed no differences in wet weight or norepinephrine or epinephrine whole tissue levels in any of the groups. As previously reported, the overall growth of intraocular septal transplants was approximately twice as great in the OX-26-NGF group relative to all other groups. This difference in final size persisted unabated for at least 5 months after the last injection. Furthermore, the significantly higher numbers of choline acetyl transferase immunoreactive neurons in transplants of OX-26-NGF-treated hosts also persisted during the 5-month postinjection interval. Taken together, the data suggest that the OX-26 conjugate may be a unique approach to permit passage of neurotrophin peptides into the brain in a biologically active form.

Synthesis, in vitro kinetics, and in vivo studies on protein conjugates of AZT: evaluation as a transport system to increase brain delivery.

Our method to increase the delivery of polar drugs to the central nervous system is via drug-protein conjugates with proteins that interact with and cross brain capillary endothelial cells. As a model for drugs containing a reactive hydroxyl group, AZT was conjugated via a succinate linker to two such protein carriers, the highly cationic histone H1 and an anti-transferrin receptor antibody, OX-26. The protein carriers were selected on the basis of their ability to interact with brain capillary endothelial cells by absorptive or receptor-mediated events, respectively. An in vitro pH profile of the rate of AZT release indicated that the observed hydrolysis proceeds by a specific base-catalysis mechanism. At 37 degrees C, the release of AZT proceeded at a rate approximately 10-fold faster (Kobs approximately 8 x 10(-4) min-1) than expected for a simple ester (AZT succinate; Kobs approximately 1.25 x 10(-4) min-1). Using simple model systems, product analysis revealed that intramolecular cyclization of the succinate linker accounts for the observed rate enhancement. Drug delivery in vivo was assessed using immunohistochemical techniques and quantitative brain uptake measurements with singly and doubly labeled AZT-OX-26 conjugates. Immunohistochemical staining of brain sections showed the colocalization of AZT and OX-26 in the brain vasculature. Therefore, drug can be linked to the antibody without affecting the targeting property of the antibody. Furthermore, an in vivo time course using radiolabeled conjugate showed that AZT is delivered to the brain capillaries but is not transported into the brain parenchyma with the antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood-brain barrier penetration and in vivo activity of an NGF conjugate.

Nerve growth factor (NGF) is essential for the survival of both peripheral ganglion cells and central cholinergic neurons of the basal forebrain. The accelerated loss of central cholinergic neurons during Alzheimer's disease may be a determinant of dementia in these patients and may therefore suggest a therapeutic role for NGF. However, NGF does not significantly penetrate the blood-brain barrier, which makes its clinical utility dependent on invasive neurosurgical procedures. When conjugated to an antibody to the transferrin receptor, however, NGF crossed the blood-brain barrier after peripheral injection. This conjugated NGF increased the survival of both cholinergic and noncholinergic neurons of the medial septal nucleus that had been transplanted into the anterior chamber of the rat eye. This approach may prove useful for the treatment of Alzheimer's disease and other neurological disorders that are amenable to treatment by proteins that do not readily cross the blood-brain barrier.

Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier.

Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. We have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration (Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C. & Mason, D. Y. (1984) Nature (London) 312, 162-163), labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate approximately 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.