(Verbatim from the application): In contrast to the intestine and kidney, little is known about the cellular and molecular mechanisms of peptide and peptidomimetic transport between blood and brain. This is unfortunate since the presence of tight junctions between the endothelial cella that form the blood-brain barrier (BBB) and the choroid plexus epithelial cells that form the blood-cerebrospinal fluid barrier (BCSFB) limit paracellular movement. Thus, specific transporters are required for the transcellular transport of hydrophilic compounds whether for the movement of nutrients into the brain or toxins out of the brain. The presence of peptide transporters within the brain (i.e., PEPT2 and PHT1) has generated considerable interest as to their precise anatomical location, role in neuropeptide homeostasis, significance in substrate trafficking, and potential as a drug delivery system through the blood brain and/or CSF barriers. Novel findings in our laboratory have established the functional and molecular presence of a high-affinity peptide transporter, PEPT2, in whole tissue rat choroid plexus. These preliminary results suggest that PEPT2 may play an important role in the uptake of peptides which function as neuromodulators, the clearance of degraded neuropeptides, and the efflux of some cephalosporin drugs from choroidal epithelium. Our working hypothesis is that PEPT2 is expressed in apical membranes of the choroid plexus and functions as the primary efflux pump in removing neuropeptide fragments and peptide-like drugs from cerebrospinal fluid. To test this hypothesis, the following specific aims are proposed:
Aim 1. To define the functional characteristics of peptide-mediated transport in rat choroid plexus epithelial cells in primary culture;
Aim 2. To determine the tissue distribution and membrane localization of specific oligopeptide transporters in mammalian brain;
Aim 3. To develop and validate a mouse model in which the gene encoding PEPT2 has been ablated by targeted gene disruption. The long-term objectives of this competing renewal application are to define the cellular and molecular mechanisms involved in the transport of peptides and peptide-like drugs in choroid plexus. Combined with immunoloclization experiments and studies in PEPT2-deficient mice, the proposed studies will greatly advance our understanding of the role, significance and vectorial transport of peptide substrates by PEPT2 in brain (as compared to PHT1 and other potential transporters). Moreover, in addressing fundamental questions of peptide transporter activity, expression and significance, the proposed studies may have important implications for the treatment of CNS disorders (e.g., Alzheimer's disease, AIDS dementia, stroke, epilepsy, schizophrenia and cancer) and for providing new strategies in drug design, delivery and targeting to the brain.
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