Direct visualization of peptide uptake activity in the central nervous system of the rat

Neuroscience Letters 364 (2004) 32–36

Direct visualization of peptide uptake activity in the central nervous system of the rat David A. Groneberg a,∗ , Isabel Rubio-Aliaga b , Monika Nickolaus b , Frank Döring b , Axel Fischer a , Hannelore Daniel b a

b

Biomedical Research Center, Otto-Heubner-Centre, Charité School of Medicine, Free University Berlin and Humboldt-University, Augustenburger Platz 1 OR1, D-13353 Berlin, Germany Molecular Nutrition Unit, Institute of Nutritional Sciences, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany Received 17 February 2004; received in revised form 6 April 2004; accepted 7 April 2004

Abstract Carrier-mediated transport of small peptides and peptidomimetics offers the opportunity for a targeted drug delivery across cell membranes in the central nervous system (CNS). This process is mediated by the proton-coupled transporter PEPT2 which is expressed in glial and choroid plexus cells. In the present studies, an uptake assay was established to visualize directly peptide uptake in intact rat brain slices. Accumulation of a reporter molecule, the fluorophore-labeled dipeptide derivative d-Ala-l-Lys-AMCA, was found in plexus choroideus and glial cells and uptake was inhibited by prototypical PEPT2 substrates, such as glycyl-l-glutamine and cefadroxil. The presence of PEPT2 was confirmed by RT-PCR and Northern blot analysis. This first CNS peptide and drug transport-visualizing assay may be used to examine new compounds which are carried by the proton-driven CNS peptide transporter. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Uptake; Transport; Peptide; Drug; Plexus choroideus

Transmembrane transport of peptides and peptidomimetics into cells represents an important process to meet the nutritional needs of tissues but also allows the cell-specific delivery of drugs. These transport processes, when located at the plexus choroideus may provide a route for delivery of neuropharmacologically active compounds. In the past years, oligopeptide transport activity has been described in choroid plexus and glial cells [4,23]. The underlying transporter protein is PEPT2, which was originally cloned from kidney and which shows structural similarity to the gene of the intestinal peptide transporter PEPT1 [16]. The PEPT2 protein possesses 12 membrane spanning domains and shows an identity of 47% to PEPT1 at the amino acid level. In kidney, PEPT2 is responsible for conservation of amino acid nitrogen by reabsorption of filtered short chain peptides [16]. PEPT2 is not only expressed in peripheral organs, such as the kidney [8], lung [10], mammary gland [9], but also in central and peripheral nervous system tissues ∗ Corresponding author. Tel.: +49-30-450-559-055; fax: +49-30-450-559-951. E-mail address: [email protected] (D.A. Groneberg).

[1,4,7]. PEPT2 transports a large variety of substrates. Some of these substrates have also been characterized for brain transport via PEPT2 [13,20,21] and the potential of PEPT2 for neuropharmacological drug design has been raised. The present study was undertaken to establish a new technique of in situ visualization of PEPT2-mediated transport activity which could allow new potential central nervous system (CNS) relevant substrates to be screened for uptake. Adult Sprague–Dawley rats (300–500 g body weight) were allowed free access to standard laboratory chow and tap water. For each of the experiments, tissue samples of six animals were used. All animal experiments were performed with the approval of the governmental Animal Care Committee and followed international standards for the use of animals in experimental research. For visualization of PEPT2-mediated substrate uptake, fresh ex vivo rat brain slices were employed. As reporter molecule, the fluorescent dipeptide derivative d-Ala-l-Lys-AMCA was used which was identified earlier as a specific peptide transporter substrate in cell cultures [2,15]. For uptake assays, rats were killed by CO2 inhalation. The brain was then rapidly removed, transferred to a

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D.A. Groneberg et al. / Neuroscience Letters 364 (2004) 32–36

vibratome chamber containing minimal essential medium 21011 (MEM; GIBCO, Karlsruhe, Germany) at 4 ◦ C and cut to slices of 200 ␮m (Vibratome: Leica VT 1000S). Sections were then incubated with MEM at 37 ◦ C, gassed with 95% O2 /5% CO2 . Uptake experiments were carried out by adding 25 ␮M d-Ala-l-Lys-AMCA (Fig. 1a) or alternatively D-Ala-L-Lys-AMCA and 1 mM of unlabeled competitors, such as the dipeptide glycyl-l-glutamine (Fig. 1b) and the aminocephalosporin cefadroxil (Fig. 1c). A large number of previous studies has demonstrated the specific transport of these compounds by the peptide transporters PEPT2 and PEPT1 [2,6,8,10,15,24]. Since PEPT1 is not expressed in the CNS as shown by functional studies, RT-PCR, immunoblot analyses and immunofluorescent confocal microscopy [3,4,13,19], these substrates are specifically transported by PEPT2 in this organ. Negative controls included the omission of the labeled and unlabelled substrates. Incubation was stopped after 20 min to 1 h by rinsing in MEM for 2 × 10 min. The slices were mounted on glass slides and immediately examined or fixed in 4% paraformaldehyde (in 0.1 M phosphate buffer pH 7.4) for 30 min. Fixed tissues were rinsed in phosphate buffered saline (PBS, pH

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7.4), transferred to PBS containing 18% sucrose, frozen and cut to 5 ␮m cryostat sections and examined. Incubation of 200 ␮m thin rat brain slices with 25 ␮M d-Ala-l-Lys-AMCA revealed abundant uptake of the reporter molecule and intracellular accumulation of AMCA fluorescence in different cell types (Fig. 1). High-power microscopy allowed to identify the cellular localization of fluorescence uptake activity: d-Ala-l-Lys-AMCA was transported into plexus choroideus cells and into glial cells (Fig. 1d and e). A maximal accumulation of fluorescence in both cell types was obtained 20 min to 1 h after administration of the reporter molecule. The signals were obtained equally strong in quenched and unquenched slices as well as in paraformaldehyde fixed 5 ␮m cryostat sections (data not shown). Other structures than choroid plexus or glial cells, such as vascular or neuronal cells, did not display uptake activity. When the uptake assays were performed with an addition of 1 mM unlabeled glycyl-l-glutamine, the fluorescence signals in glial and choroid plexus epithelial cells were reduced to a minimal extent (Fig. 1f). Also, incubations performed in the presence of 1 mM unlabeled cefadroxil almost

Fig. 1. Visualizing ex vivo peptide uptake. As reporter molecule and unlabeled competitive substrates, the fluorophore-labeled dipeptide derivative d-Ala-l-Lys-AMCA (a), glycyl-l-glutamine (b) and cefadroxil (c) were used. Incubations of 200 ␮m vibratome slices with 25 ␮M d-Ala-l-Lys-AMCA lead to an uptake that was restricted to choroidal epithelial cells (d) and glial cells (e) which was inhibited when higher concentrations of unlabeled glycyl-l-glutamine (f) and cefadroxil (g) were added to the incubation solutions. Scale bar represents 25 ␮m (e–g), 105 ␮m (d).

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abolished cellular accumulation of fluorescence (Fig. 1g). Control incubations without labeled and unlabeled substrates were completely unstained (data not shown). For validating PEPT2 expression, RT-PCR studies were performed as previously described [12]. In brief, a PEPT2specific reverse primer (5 -ACCATGCCTTCAGCAGCCTCT-3 ) was used to generate a single stranded cDNA by reverse transcription. PCR was performed using the reverse and a forward primer (5 -GCTGCCTACTGAAGCCAAATGCTTG-3 ). After an initial denaturation step (5 min at 94 ◦ C) Taq-polymerase was added and 30 cycles (1 min at 94 ◦ C, 1 min at 55 ◦ C, 1 min at 72 ◦ C) were performed followed by end synthesis (10 min at 72 ◦ C). A 1/10 volume of each sample was separated on a 1% agarose gel and stained by ethidium bromide. PCR controls were performed by using H2 O instead of cDNA. Using mRNA from rat brain and kidney as control organ, PEPT2-specific amplification products with a length of 341 bp were detected in both organs (Fig. 2). Expression of the housekeeping gene GAPDH was positive in both kidney and brain probes, giving a specific amplification product of 452 bp (Fig. 2). PEPT2 mRNA was also identified by Northern blot studies using a rat-specific digoxigenin-labeled PEPT2 probe. Northern blot analysis was performed as previously described [11] by using DIG-labeled PEPT2 antisense and sense probes. In brief, a rat-specific PEPT2 PCR fragment (nucleotides 51–290) was cloned into the pCRII vector (Invitrogen, Leck, The Netherlands). Plasmids were linearized by restriction enzymes (NotI/BamHI), phenol–chloroform extracted, EtOH-precipitated and transcribed by T7 RNA polymerase or Sp6 RNA polymerase, respectively. 10 ␮g of total or poly(A)+ RNA (2 ␮g) prepared from rat brain was

separated by agarose gel electrophoresis and transferred onto nylon membranes (Boehringer, Mannheim). Lanes were hybridized with digoxigenin-labeled PEPT2-specific cRNA probes. Hybridization was carried out overnight at 65 ◦ C in the presence of 50% deionized formamide, 5× SSC, 0.1% (w/v) N-lauroylsarcosine, 0.02% (w/v) SDS and 2% blocking reagent. After hybridization, membranes were washed twice for 15 min at 65 ◦ C in 2× SSC containing 0.1% SDS and twice for 15 min at 65 ◦ C in 0.5× SSC containing 0.1% SDS. Detection of the DIG-labeled hybrids was carried out according to the manufacturer’s digoxigenin detection kit for glycoconjugate and protein analysis protocol (Boehringer Mannheim, Mannheim, Germany; developing times 2–4 h). The hybridization signal with a size of 4.2 kb obtained from brain RNA was identical to the signal obtained in the control rat kidneys (Fig. 2). No hybridization signal was obtained using the sense probe (data not shown). The choroid plexus is responsible for the homeostatic control of the composition of the cerebrospinal fluid (CSF) which is accomplished by the regulation of multiple transport systems allowing a balanced pH, ionic, neurotransmitter and nutrient concentration. It has been previously demonstrated that the Na+ -independent but proton-gradient driven peptide symporter PEPT2 is functionally expressed in choroid plexus epithelial cells and, therefore, a nutritional and regulatory role was attributed to PEPT2 [20]. By its capability to transport a variety of peptidomimetics, PEPT2 was discussed as a delivery system in new strategies of drug design and targeting to the brain [20]. The present studies were carried out to establish a visualization assay for peptide and peptidomimetic transport in

Fig. 2. Detection of PEPT2 mRNA in rat brain by RT-PCR and Northern blotting. 5 ␮g of total RNA from rat kidney and rat brain was submitted to reverse transcription and amplification with rat PEPT2-specific primers. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. DNA marker: Lambda DNA-BstE II. Samples of total RNA (10 ␮g) and poly(A)+ RNA (2 ␮g) from rat kidney and brain were separated by agarose gel electrophoresis, blotted and hybridized with a specific PEPT2 antisense probe. RNA marker: Boehringer Mannheim.

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the CNS, which may be used in future drug delivery studies. Utilizing this approach to directly visualize peptide uptake in brain tissues, PEPT2 activity in cells is easily assessed by reporter molecule uptake. Previous studies on the minimal molecular requirements in substrates for transport by PEPT2 showed the importance of a free N-terminal amino group and a free carboxy terminus, whereas the side chains in peptides allow introduction of rather large groups [22]. This allowed the reporter molecule d-Ala-l-Lys-AMCA to be synthesized for assessing transport with increased enzymatic stability by incorporation of the d-Ala residue into the amino terminus. This fluorescent dipeptide conjugate has also been demonstrated to represent a useful transporter substrate in cell culture systems [2,15]. The specificity of the d-Ala-l-Lys-AMCA-based CNS uptake assay with an activity found in glial and choroid plexus cells was verified by competitive inhibition studies and a variety of controls. Firstly, it was shown that the transport of d-Ala-l-Lys-AMCA can be inhibited by high concentrations of the aminocephalosporin cefadroxil and the dipeptide glycyl-l-glutamine. Both molecules have been earlier characterized as competitive acting peptide transporter substrates [6]. Secondly, a major passive uptake of the reporter molecule by diffusion was excluded by lack of staining in surrounding structures, such as vascular or neuronal cells. Thirdly, the incorporation of the d-Ala residue to render the reporter molecule more hydrolysis-resistant prevented generation and uptake of fluorescent fragments. To verify the presence of PEPT2 in the presently examined tissues and cells in CNS of Sprague–Dawley rats, consecutive RT-PCR and Northern blotting experiments were performed with brain and control kidney extracts which confirmed PEPT2 mRNA expression. The cell-type-specific accumulation of d-Ala-l-LysAMCA in plexus choroideus cells and glial cell is in accordance with an earlier report on the distribution of PEPT2 mRNA in the central nervous system [1] and extends these finding by the first direct in situ visualization of uptake activity in these cells. Functional studies performed in synaptosomes prepared from rat also indicated that PEPT2 is expressed in the cerebral cortex [5]. Recently, a targeted PEPT2 gene deletion in mice was examined with regard to peptide uptake in the kidney [17] and in the choroid plexus [18]. It was shown that the proton-dependent uptake of glycylsarcosine by PEPT2 in isolated choroid plexus cells was markedly impaired providing further strong evidence that PEPT2 is the primary peptide transporter involved in choroidal peptide uptake [18]. Similar findings were obtained for choroidal cefadroxil uptake [14]. Recently, it was demonstrated that PEPT2 is expressed only at the apical surface of choroid plexus cells and that transport mainly occurs from the apical to the basal side of the epithelium. This implies that the favored direction of peptide transport is from CSF to blood, not vice versa [19]. In conclusion, the present study reports a new ex vivo uptake assay for PEPT2-mediated transport in the central

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nervous system which combines the ease of visualization of carrier-mediated efficient uptake of a specific reporter molecule and its competitive substrates with the conserved functional morphology of the tissue using intact brain slices and vibratome technique. This uptake assay visualizes for the first time PEPT2 activity in intact, living brain slices with the possibility to distinguish between different cell types and may be particularly useful for future approaches in rational drug design for transport and targeting studies in the central nervous system.

Acknowledgements We thank R. Strozynski for skilful technical assistance. Source of support: Deutsche Nierenstiftung.

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