Human brain samples
This study was approved by the ethics committee of Universidad Miguel Hernández de Elche, Spain, and it was carried out in accordance with the WMA Declaration of Helsinki. Brain samples (frontal cortex; see Table 1) were obtained from the Brain Bank of the Institute of Neuropathology, Bellvitge University Hospital. Cases with AD-related pathology were considered those showing neurofibrillary tangles (NFT) and/or senile plaques with the distribution established by Braak and Braak at the post-mortem neuropathological examination [18]. These were categorized as Braak NFT stages I–II n = 14, 1 female/13 males, 68.4 ± 8.8 years; Braak stages III–IV, n = 14, 7 females/7 males, 80.4 ± 8.2 years; and Braak stages V–VI, n = 12, 5 females/7 males, 76.5 ± 9.7 years. Cases at NFT stages I–II showed no or moderate numbers of senile plaques (mostly scores 0 and A); cases at stages III–IV usually had moderate numbers of senile plaques (mostly score B); cases at stages V–VI had heavy senile plaque burden (mostly score C; Table 1). Cases at stages I, II, and III did not have cognitive impairment; three cases at stage IV had moderate cognitive impairment, and cases at stages V and VI had suffered from dementia. Special care was taken not to include cases with combined pathologies to avoid bias in the pathological series. Samples from middle-aged (MA) subjects (3 females/8 males; average age 51.8 ± 4.8 years) corresponded to individuals with no neurological diseases and no evidence of NFTs and senile plaques. The mean post-mortem interval of the tissue was ~8 h in all cases, with no significant difference between the groups.
A major concern in the design of the study is the age of the different groups of human cases. MA individuals are younger (51.8 ± 4.8 years) when compared with cases with AD-related pathology (NFT I–II 68.4 ± 8.8, III–IV 80.4 ± 8.2, and V–VI 76.5 ± 9.7). This selection is due to the fact that the majority of individuals aged 65 years or older have stages I–III of NFT pathology, and, therefore, it is difficult to have samples of age-matched controls without AD-related pathology and morbidities considered in the selection of NFT series that could have an impact on the results [20].
Cell cultures
SH-SY5Y cells, a human neuroblastoma cell line, were seeded at a density of 1×105 cells/well in 6-well plates and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with Glutamax (GIBCO Thermo Fisher Scientific, Rockford, USA), 1% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) in a 5% CO2 incubator. To neuro-differentiate the cells, all-trans-retinoic acid (RA, Sigma-Aldrich Co, MO, USA) was employed. RA enhances neuronal markers, reelin and ApoER2 expression [21, 22]. Ten micromolar RA diluted in DMEM with 1% FBS was added every 2 days. After 6 days, cells were treated with recombinant reelin, 12 μg/ml for 24 h. Other cells were treated with suspensions of β-amyloid 1–42 (Aβ42) or scrambled control peptide (Aβsc; AIAEGDSHVLKEGAYMEIFDVQGHVFGGKIFRVVDLGSHNVA) (both from Anaspec Peptide, Eurogentec) in DMEM with 1% FBS, for two consecutive days without changing the media, at a final concentration of 500 nM, 1 μM, or 5 μM.
Non-differentiated SH-SY5Y cells were transfected with Lipofectamine 3000 (ThermoFisher) following manufacturer’s instructions, with a construct encoding full-length ApoER2 (pEGFPN1-Mus musculus ApoER2, residues 1–842) and ApoER2-ICD-HA expressing only the cytoplasmic domain (residues 728–842) (both generously provided by Dr W. Rebeck; see ref. [23, 24]), or with GFP/cDNA3.1 as mock transfection as in [14] for 48 h. After 24 h post-transfection, some CHO-PS70 cells were treated with 10 μM chloroquine for another 24 h.
CHO cells stably overexpressing wild-type human APP (CHO-PS70, [25]) were grown in DMEM® containing 10% FBS, 0.1% Puromycin (Sigma-Aldrich), and 0.2% G418 disulfate salt (Sigma-Aldrich). CHO-PS70 cells were transfected with full-length human LRP3 cDNA (3×FLAG-LRP3 in pCMV7.1; a kind gift from Christine Lavoie, [26]) for 48 h. After 24 h post-transfection, some CHO-PS70 cells were treated with 10 μM chloroquine for 24 h.
Brain membrane-enriched fractions
Brain cortex samples were homogenized using a polytron Heidolph RZR-1 at 600–800 rpm, in a glass potter applying 10–15 pulses in buffer at 10% (w/v) (Hepes 1mM, sucrose 0,32 M, Cl2Mg mM, EDTA 1mM, NaHCO3 1mM, PMSF, protease inhibitors (Cocktail Complete EDTA free, Roche), antiphosphatase inhibitor (PhosSTOP, Sigma)). The homogenate was centrifuged at 1000 ×g during 20 min at 4°C. The supernatant (post-nuclear fraction) was centrifuged at 13000 ×g during 15 min at 4°C. The supernatant (cytosolic fraction) was aliquoted, and the resulting pellet (membrane-enriched fraction) was resuspended in buffer (Hepes 1mM, Cl2Mg mM, EDTA 1mM, NaHCO3 1mM, PMSF, protease inhibitor cocktail (Sigma-Aldrich), antiphosphatase inhibitor (Sigma-Aldrich)).
In some CHO-PS70 cells, we performed a differential centrifugation. After homogenization of cell extracts in sucrose buffer (0.32 M sucrose, 10 mM Tris pH 7.4, EGTA, 1 mM Na3VO4, 5 mM NaF, 1 mM EDTA, 1 mM Hepes), the homogenate was centrifuged at 1000 ×g for 10 min. The supernatant was centrifuged at 15000 ×g for 15 min. The resultant supernatant (fraction containing mainly the plasma membrane and soluble proteins from the cytosol) and the pellet (containing mainly membranes from the endoplasmic reticulum, mitochondria, lysosomes, peroxisomes, and endosomes) were quantified and stored.
Microarray analysis
Gene expression was analyzed 48 h after transfection with human full-length ApoER2, using microarrays SurePrint G3 Human Microarrays (ID 039494, Agilent Technologies, Spain) and performed by Bioarray SL (http://www.bioarray.es). The concentration and purity of the total RNA extracted were measured by a NanoDrop spectrophotometer, and RNA quality was determined with the kit R6K Screen Tape (Agilent Technologies, Spain). The estimated RNA integrity number ranged between 9.5 and 9.7. Each sample (four samples and four controls) was labeled with Cy3 using the One-Color Microarray-Based Gene Expression Microarrays Analysis v.6.6 (Agilent Technologies, Spain). Data were imported to the linear models for microarray data Bioconductor software (Limma, Marray, affy, pcaMethods and EMA). Raw data were first subjected to background subtraction, then to within-array loess normalization. Finally, across-array normalization was performed. Normalized data were fitted to a linear model. The significance of the gene expression changes was analyzed according to the adjusted p value (adj. p < 0.05).
qRT-PCR analysis
RNA was extracted from human brains, SH-SY5Y cells, or CHO-PS70 cells using the TRIzol® Reagent in the PureLink™ Micro-to-Midi Total RNA Purification System (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. SuperScript™ III Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) was used to synthesize cDNAs from this total RNA (2 μg) using random primers according to the manufacturer’s instructions. Quantitative PCR amplification was performed on a StepOne™ Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Rockford, USA) with TaqMan probes specific for human LRP3 (assay ID: HS01041220_m1), LDLR (assay ID: HS00181192_m1) (Applied Biosystems, Thermo Fisher Scientific, Rockford, USA), and human 18S as a housekeeping gene (Applied Biosystems, Thermo Fisher Scientific, Rockford, USA) for the human brain and SH-SY5Y cell samples. In CHO-PS70, mRNA expression was measured with primers for human APP (forward: AACCAGTGACCATCCAGAAC; reverse: ACTTGTCAGGAACGAGAAGG) and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, forward: AGAAGGTGGTGAAGCAGGCAT; reverse: AGGTCCACCACTCTGTTGCTGT) to normalize the expression levels of the target gene by the ΔCt method curves.
APOE genotyping was performed by qRT-PCR according to a previously described method [27].
Recombinant reelin
HEK-293T cells stably transfected with full-length mouse reelin clone pCrl or GFP (mock) (kindly provided by Dr. E. Soriano, Department of Cell Biology, University of Barcelona, Barcelona, Spain) were seeded in 175-cm2 flasks at a density of 10×106 cells/flask. After 3 days in culture in Optimem, the supernatants were filtered through 0.2-μm pores and concentrated with an Amicon Ultra 100-kDa size exclusion filter (Merk Millipore, Darmstadt, Germany). For quantification, a coomasie gel was loaded with different volumes of the concentrated supernatants as well as with different bovine serum albumin solutions to perform an extrapolation.
Western blotting
Brain membrane-enriched fractions, SH-SY5Y extracts, or CHO-PS70 extracts (30 μg) were run on SDS-PAGE (7.5%, 12%, precast 4–15% gradient, or Tris-tricine 16%) after boiling at 98°C for 5 min in 6× Laemmli sample buffer. Proteins were transferred by electrophoresis to nitrocellulose membranes and detected with antibodies against the C-terminal of LRP3 (mouse, 1:100, Sigma-Aldrich, St. Louis, MO, USA), N-terminal of LRP3 (rabbit, 1:100, Sigma-Aldrich), Flag (mouse, 1:1000, Sigma-Aldrich), C-terminal of LDLR (rabbit, 1:200, Sigma-Aldrich), C-terminal of ApoER2 (rabbit, 1: 2000, Abcam, Cambridge, UK), C-terminal of APP (rabbit, 1: 2000, Sigma-Aldrich), N-terminal of APP (rabbit, 1: 2000, Sigma-Aldrich), sAPPα (mouse, 1:1000; IBL, Hamburg, Germany), sAPPβ (rabbit 1:1000; IBL), LC3B (rabbit, 1:2000; Abcam), or α-tubulin (1:4000, Sigma-Aldrich) as a loading control. Primary antibody binding was visualized with fluorescent secondary antibodies (IRDye, 1: 10000), and images were acquired using an Odyssey CLx Infrared Imaging system (LI-COR Biosciences GmbH). Representative whole blots are shown as Supp Fig. 1.
Immunoprecipitation
Brain extracts (100 μL) or CHO-PS70 extracts (50 μL) were incubated on a roller for 2.5 h at room temperature with 100 μL of magnetic beads (Dynabeads, Merck Millipore) coupled to the C-terminal LRP3 (mouse, Sigma-Aldrich) for brain extracts, C-terminal APP (rabbit, Biolegend) for CHO-PS170 extracts, or mouse/rabbit IgG (negative controls). The input, bound, and unbound fractions were analyzed by western blotting using specific antibodies.
Immunofluorescence
CHO-PS70 cells overexpressing LRP3-flag were washed with cold Hank-buffered salt solution and fixed with 4% paraformaldehyde and 0.1 M EGTA for 10 min. To stain the plasma membrane, cells were incubated with WGA-FITC (WGA: lectin from Triticum vulgaris, FITC (fluorescein) conjugate, Sigma-Aldrich) for 15 min at room temperature, and the nonspecific sites were blocked with 10% (w/v) bovine serum albumin for 30 min. No permeabilization steps were included before or during the incubation with the primary antibodies. Cells were incubated with a primary antibody against Flag (1:200; mouse; Sigma-Aldrich) for 1 h, followed by the secondary antibody (1:200, Cy5 anti-mouse; GE-Healthcare) for 1 h. After washes with PBS, cells were incubated briefly with Hoechst dye to label nuclei (Invitrogen). Pictures were acquired in a Leica SPEII upright TCL-SL confocal microscope using an oil-immersion 40× objective
Double-labeling immunofluorescence and confocal microscopy
The frontal cortex and hippocampus of 14 cases at Braak NFT stages 0–I, IV, and V–VI and senile plaque stages 0–C were used in the study. Formalin-fixed, paraffin-embedded, de-waxed sections, 4 μm thick, were stained with a saturated solution of Sudan black B (Merck) for 15 min to block autofluorescence of lipofuscin granules present in cell bodies and then rinsed in 70% ethanol and washed in distilled water. The sections were boiled in citrate buffer to enhance antigenicity and blocked for 30 min at room temperature with 10% fetal bovine serum diluted in PBS. Then, the sections were incubated at 4°C overnight with combinations of primary antibodies: LRP3-C-term (Sigma-Aldrich, ref SAB1300316, polyclonal rabbit, diluted at 1:50) and apoER2 (Invitrogen, ref MA5-36130, mouse monoclonal, diluted 1:50). After washing, the sections were incubated with Alexa488 or Alexa546 (1:400, Molecular Probes) fluorescent secondary antibodies against the corresponding host species. Nuclei were stained with DRAQ5TM (1:2000, Biostatus). After washing, the sections were mounted in an Immuno-Fluore mounting medium (ICN Biomedicals), sealed, and dried overnight. Sections were examined with a Leica TCS-SL confocal microscope.
Statistical analysis
The distribution of data was tested for normality using a D’Agostino-Pearson test. ANOVA was used for parametric variables and the Kruskal-Wallis test for non-parametric variables for comparison between groups. A Student’s t-test for parametric variables and a Mann-Whitney U test for non-parametric variables were employed for comparison between two groups and for determining p values. For data analyzed using unpaired Student’s t-test, a Welch’s correction was employed in data with different standard deviations. Correlation between variables was assessed by linear regression analyses. The results are presented as the means ± SE, and all the analyses were performed using GraphPad Prism (version 7; GraphPad Software, Inc). p value < 0.05 was considered significant.