Chemicals and assay kits were purchased from the following sources: FAM-labeled Aβ (catalog # 23514-01) from Anaspec (San Jose, CA), SOD assay kit (catalog # K335-100) from Biovision (Mountain View, CA), ELISA kit (catalog # KHB-3481) and ultra purified Aβ (1–40) (catalog # 03-138) from Biosource (Carlsbad, CA), rabbit anti LRP1 antibody (catalog # ARP32793) from Aviva (San Diego, CA), Alexa-labeled secondary antibody from Molecular Probes (Eugene, OR), enhanced chemiluminescene reagent (ECL) and ECL films from Amersham Biosciences (Piscataway, NJ), Dulbecco’s modified essential medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin, gentamycin from Gibco (Grand Island, NY), the PCR buffer, dNTP, Oligo dT and MuLV reverse transcriptase from Applied Biosystems (Foster City, CA), LDH assay kit (catalog # TOX-7), β-actin, dithiothreitol (DTT), 2-mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), polyacrylamide, tetramethyl-ethylenediamine (TEMED), and the siRNA for LRP1 and all other chemicals were from Sigma Chemicals (St. Louis, MO). TRIzol was purchased from Invitrogen (Carlsbad, CA), oligodT, MuLV from Applied Biosystems (Foster City, CA), the ABsolute QPCR SYBR green Mix kit from ABgene (Rochester, New York), the primers from Integrated DNA Technology Inc. (Coralville, IA), and transfection agent, lipofectamine from Ambion (Austin, TX). All reagents were of analytical grade, HPLC grade or the best available pharmaceutical grade.
Animals and treatment
Male Sprague-Dawley rats at the time they were used were 8–9 weeks old (250–300g). The animals were housed in a temperature-controlled, 12:12 light/dark room, and were allowed free access to tap water and food. Rats received an ip injection of 50 mg/kg Pb acetate (i.e., 27 mg Pb/kg) or an equivalent molar concentration of Na-acetate (i.e., 15 mg acetate/kg) as controls. Twenty-four hours post injection the rats were anesthetized with ketamine/xylazine (75:10 mg/mL, 1 mL/kg body weight), immobilized in a stereotaxic device and subjected to the following experimentation. Animal protocols pertinent to this study were approved by the Purdue University Animal Care and Use Committee.
Intraventricular perfusion of Aβ
A midline cutaneous incision was made from the head to the neck on the dorsal surface to expose the skull. A hole was drilled in the skull at co-ordinates determined using the Paxinos and Watson Atlas at 0.8 mm posterior to bregma and 1.4 mm lateral) followed by an insertion of a sterilized cannula at 3.5 mm ventral from the skull surface. An internal cannula connected to PE 50 tubing was inserted into the guide cannula for lateral ventricle perfusion. The other end of the PE tubing was attached to a 10 μL Hamilton syringe, which was filled with 200 pmoles of FAM-labeled Aβ diluted in artificial CSF (Yamada et al., 1998).
The Aβ solution was infused into the lateral ventricle at a rate of 12 μL/min for 0.5 min. The cannula was allowed to remain inside the ventricle for an additional 0.5 min before it was removed in order to avoid the back flow into the tubing. Twenty min post infusion, rats were euthanized with ketamine-xylazine and the brain was dissected to remove the choroid plexus. The time of Aβ incubation (15–20 minutes) was based on previous studies, which establishes a clearance of Aβ by the BBB within 30 minutes (Bell et al., 2007
). The tissues were then transferred to a 35-mm dish and washed three times with an artificial CSF (aCSF). Aliquots (1–2 drops) of aCSF were then added on the tissue to prevent it from drying out; the tissue was observed immediately using an inverted laser scanning microscope (Olympus, FV1000) for live uptake.
Confocal immunofluorescence microscopy
To acquire images, the 35mm dish containing the choroid plexus specimen in artificial CSF was mounted on the stage of an Olympus, FV1000 inverted confocal laser-scanning microscope and viewed through a 40x water-immersion objective (numeric aperture=1.2), with a 488-nm laser line for excitation (Ar-ion laser). Low laser intensity was used to avoid photo bleaching. The choroid plexus was examined under reduced transmitted-light illumination and an area containing undamaged epithelium with underlying vasculature was selected. Each sample was imaged at a rate of one frame per second and care was taken to expose all the tissues for the same period of time at a reduced illumination setting to avoid photo bleaching. Confocal images (512×512×8 bits, 4 frames averaged) were acquired and saved to a disk. For each tissue sample, 4 areas of cells were selected for image collection.
The fluorescence intensity was further quantified using software ImageJ and reported in arbitrary units (a.u). Data reported, unless otherwise stated, are the results of single experiments representative of three to four replicate experiments.
Culture of choroidal epithelial Z310 cells
The characteristics of immortalized rat choroidal epithelial Z310 cells have been described in a previous publication (Zheng and Zhao, 2002
). Briefly, cells were maintained in DMEM (high glucose) medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 40μg/mL of gentamycin in a humidified incubator with 95% air–5% CO2
at 37°C and were passaged twice a week.
Determination of Pb-induced cellular toxicity in Z310 cells
To choose the Pb concentration at which it altered Aβ transport by the choroid plexus but did not induce nonspecific cytotoxicity, three general cytotoxicity assays were used, including the methylthiazolyldiphenyl-tetrazolium bromide cell viability assay (MTT), cell membrane permeability assessment (lactate dehydrogenase or LDH assay), and cellular oxidative stress estimation (superoxide dismutase or SOD assay). MTT cell viability assay was performed by growing Z310 cells at a density of 40,000 cells/well for 2–3 days until they reached 80–85% confluence. The medium was then removed and replaced with fresh medium containing different concentrations of Pb as Pb-acetate (0, 5,10, 25, and 50 μM). The cells were incubated for an additional 24 h, followed by adding an aliquot of MTT stock solution (2 mg/mL in PBS) to each well. The absorbance of the converted dye was measured at a wavelength of 570 nm. To determine the LDH activity, Z310 cells were treated in the same way as described in MTT assays. An LDH assay was then conducted using a LDH assay kit as per protocol. The SOD activity was determined according to the instruction of the assay kit. The cells were treated with Pb at 0 or 10 μM for 24 h.
Detection of Aβ1–40 in Z310 Cells following Pb exposure by immunofluorescence
The effects of Pb exposure on Aβ uptake in choroidal epithelial cells were qualitatively assessed using confocal microscopy. Z310 cells were plated on 35-mm glass plates (MatTek, Ashland, MA) and grown until they reached about 85% confluence (2–3 days). The cells were treated with 10 μM Pb as Pb acetate for 24 h in DMEM medium. Following exposure, the cells were washed 3 times with phosphate buffered saline (PBS) to remove all remnants of Pb in the dish. Cells were then incubated with 200 μL of 2 μM fluorescent labeled Aβ for one hour followed by three PBS washes. Fresh cell culture medium was then added and the preparations were observed under a confocal microscope.
Quantification of Aβ1–40 accumulation in Z310 cells following Pb exposure by ELISA
Intracellular accumulation of Aβ in Z310 cells following Pb exposure was further quantified using a well established enzyme linked immunosorbent assay (ELISA) After Pb exposure at 10 μM for 4, 12, 24 or 48 h, Z310 cells were washed with PBS and incubated with 200 μL of a 2 μM solution of unlabeled ultrapure Aβ1–40 in serum free medium for 1 h. Cells were washed 3 times with PBS to remove excess Aβ, collected and sonicated to lyse the cells. The cell lysates were diluted 3:1 with diluent buffer (as per manufacturer’s instructions) before adding them to the ELISA plates. Aβ1–40 colorimetric kit (Invitrogen KHB3481) was used to determine the concentrations of Aβ1–40 in the cell lysates. This assay kit detects monomeric Aβ1–40 since this form is more likely to be transported across the barrier rather than the aggregated form. An aliquot of cell lysates was used to quantify the protein concentrations by the Bradford assay to normalize the Aβ1–40 to total protein.
Quantification of LRP1 mRNA expression by real-time RT-PCR
The transcription of the gene encoding LRP1 was quantified using real-time RT-PCR as described by Walker et al., (2001). Briefly, total RNA was isolated from Z310 cells or choroid plexus tissue using TRIzol reagent following the manufacturer’s directions. An aliquot of RNA (1 μg) was reverse-transcribed with MuLV reverse-transcriptase and oligo dT primers. The forward and reverse primers for target genes were designed using Primer Express 3.0 software. The ABsolute QPCR SYBR green Mix kit (ABgene, Rochester, New York) was used for real-time RT-PCR analyses. The amplification was carried out in the MX 3000P real-time PCR System (Stratagene, La Jolla, CA). Amplification conditions were 15 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 1 min at 55 °C and 30 s at 72 °C. A dissociation curve was used to verify that the majority of fluorescence detected could be attributed to the labeling of specific PCR products, and to verify the absence of primer-dimers and sample contamination.
All real time RT-PCR reactions were done in triplicate. Primers sequences for rat LRP1 used for real-time RT-PCR were: forward primer 5′-TTGTGCTGAGCCAAGACATC -3′ and a reverse primer 5′GGCGTGGAAGACATGTAGGT -3′ (Genbank Accession No XM_243524) and rat glyceraldehydes-3-phosphate dehydrogenase (GAPDH), used as an internal control, had a forward primer 5′-CCT GGA GAA ACC TGC CAA GTA T-3′ and a reverse primer 5′-AGC CCA GGA TGC CCT TTA GT-3′ (Genbank Accession No. NM_017008).
Quantification of LRP1 protein expression by Western blot
The choroid plexus tissues or Z310 cells were homogenized (1:10, wt/vol) on ice in a buffer containing 20 mM Tris (pH 7.5), 5 mM EGTA, 1% TritonX-100, 0.1% SDS, 50μM phenylmethylsulphonylfluoride (PMSF), 15 mM 2-mercaptoethanol and a Protease Inhibitor Cocktail containing 500 μM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 150 nM aprotinin, 1 μM E-64, 0.5 mM EDTA, 1 μM leupeptin (Calbiochem, San Diego, CA). Samples were sonicated using a Model 500 Sonic Dismembrator (Fisher Scientific) at duty cycle 20% and output 4–6 for 30 pulses. Following centrifugation at 10,000 g at 4°C for 10 min, aliquots of supernatants were assayed for protein concentrations by the Bradford method. A volume of protein extract (40 μg of protein) was mixed with an equal volume of 2X sample buffer (0.35 M Tris-Cl, 10% SDS, 30% glycerol, 0.6 M DTT, and 0.012% bromophenol blue), loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and then transferred to a PVDF membrane. The membrane was blocked with 5% dry milk in TBST (Tris-buffered saline) at room temperature for 1 h and immunoblotted with an antibody directly against LRP1(1:250). This antibody, purchased from Aviva Systems Biology (accession number Q6PJ72), identifies the swissprot ID of LRP1 at 48 kD. The membrane was stained with a horse-radish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:5000) at room temperature for 1 h and developed using ECL reagent and films. The exposure time varied from 30 sec to several min depending on signal strength. β-actin (42 kD) (1:2000) was used as an loading control; the corresponding secondary antibody (1:2000) for β-actin was HRP-conjugated goat anti-mouse IgG. Band intensities were quantified using Scion Image software (Frederick, Maryland) and results were reported as a ratio of LRP1 to β-actin in the tissue or cells.
Aβ accumulation following LRP1 knockdown by RNAi
siRNA Transfection was performed as follows: Candidate sequences for LRP1 knockdown were obtained commercially from Sigma-Aldrich. The sequences used were- Forward primer: 5′ CCUAUCUUUGAGAUCCGAA 3′; reverse Primer: 5′ UUCGGAUCUCAAAGAUAGG 3. Transfection agent lipofectamine was found to work best in the Z310 cells after a series of screening. Transfection conditions were optimized according to the following variables” initial seeding density, volume of transfection agent, duration of transfection, concentration of siRNA.
Cells were seeded at a density of 1× 105 cells/well in a 6- well plate in cell culture medium. After 24 hours, the RNA/transfection system was prepared as follows: 1 ul lipofectamine was diluted in 100 uL OPTI-MEM 1 medium and incubated for 10 minutes at room temperature. siRNA was added to a separate 100 uL OPTI- MEM medium to obtain a final concentration of 50 nM per dish. The transfection agent and the siRNA were then mixed and incubated at room temperature for 45 minutes with occasional mixing. Cell culture medium was replaced with 200ul of the above mixture along with 800 uL of OPTI-MEM medium to obtain a total of 1 ml medium/well for 5 hours. An additional 1 ml of regular cell culture medium was added and the cells were grown for an additional 48 hours. The cells were transfected with either scrambled siRNA as a negative control or the siRNA sequence designed homologous to LRP1. A negative control was used to demonstrate that there was no non-specific toxicity caused by the transfection agent Knockdown was then analyzed by laser scanning cytometry, real time RT PCR and western blot analysis. Cells were exposed to Pb at 10 μM for 24 hours. Aβ uptake studies were performed as described earlier using ELISA and intracellular Aβ was normalized to total protein. The intracellular Aβ levels were normalized by the cellular total protein concentrations.
Statistical analyses of the differences between groups were carried out by a one-way ANOVA with post hoc comparisons by the Dunnett’s test or using paired t–tests (Kaleidagraph 3.6) and by using SPSS (version 30.0) to determine correlation coefficients All data are expressed as mean ± SD. Differences between two means were considered significant when p was equal or less than 0.05.