The apolipoprotein receptor LRP3 compromises APP levels

Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 https://doi.org/10.1186/s13195‑021‑00921‑5 RESEARCH The apolipoprotein receptor LRP3 compromises APP levels Inmaculada Cuchillo‑Ibañez1,2,3* , Matthew P. Lennol1,2, Sergio Escamilla1,2, Trinidad Mata‑Balaguer1,2, Lucía Valverde‑Vozmediano1, Inmaculada Lopez‑Font1,2,3, Isidro Ferrer2,4 and Javier Sáez‑Valero1,2,3* Abstract Background: Members of the low‑density lipoprotein (LDL) receptor family are involved in endocytosis and in transducing signals, but also in amyloid precursor protein (APP) processing and β‑amyloid secretion. ApoER2/LRP8 is a member of this family with key roles in synaptic plasticity in the adult brain. ApoER2 is cleaved after the binding of its ligand, the reelin protein, generating an intracellular domain (ApoER2‑ICD) that modulates reelin gene transcrip‑ tion itself. We have analyzed whether ApoER2‑ICD is able to regulate the expression of other LDL receptors, and we focused on LRP3, the most unknown member of this family. We analyzed LRP3 expression in middle‑aged individuals (MA) and in cases with Alzheimer’s disease (AD)‑related pathology, and the relation of LRP3 with APP. Methods: The effects of full‑length ApoER2 and ApoER2‑ICD overexpression on protein levels, in the presence of recombinant reelin or Aβ42 peptide, were evaluated by microarray, qRT‑PCRs, and western blots in SH‑SY5Y cells. LRP3 expression was analyzed in human frontal cortex extracts from MA subjects (mean age 51.8±4.8 years) and AD‑related pathology subjects [Braak neurofibrillary tangle stages I–II, 68.4±8.8 years; III–IV, 80.4 ± 8.8 years; V–VI, 76.5±9.7 years] by qRT‑PCRs and western blot; LRP3 interaction with other proteins was assessed by immunoprecipi‑ tation. In CHO cells overexpressing LRP3, protein levels of full‑length APP and fragments were evaluated by western blots. Chloroquine was employed to block the lysosomal/autophagy function. Results: We have identified that ApoER2 overexpression increases LRP3 expression, also after reelin stimulation of ApoER2 signaling. The same occurred following ApoER2‑ICD overexpression. In extracts from subjects with AD‑related pathology, the levels of LRP3 mRNA and protein were lower than those in MA subjects. Interestingly, LRP3 transfection in CHO‑PS70 cells induced a decrease of full‑length APP levels and APP‑CTF, particularly in the membrane fraction. In cell supernatants, levels of APP fragments from the amyloidogenic (sAPPα) or non‑amyloidogenic (sAPPβ) pathways, as well as Aβ peptides, were drastically reduced with respect to mock‑transfected cells. The inhibitor of lysosomal/ autophagy function, chloroquine, significantly increased full‑length APP, APP‑CTF, and sAPPα levels. Conclusions: ApoER2/reelin signaling regulates LRP3 expression, whose levels are affected in AD; LRP3 is involved in the regulation of APP levels. Keywords: sAPP, ApoER2, ApoER2‑ICD, Beta‑amyloid, Alzheimer’s disease, Chloroquine, Differential centrifugation, Autophagy © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction The members of the family of low-density lipoprotein (LDL) receptors are endocytic receptors that mediate the uptake of lipoproteins and have been classically studied for their role in cholesterol transport and metabolism. Robust evidence indicates that LDL receptor family Open Access *Correspondence: [email protected]; [email protected] 1 Instituto de Neurociencias de Alicante, Universidad Miguel Hernández de Elche‑CSIC, Sant Joan d’Alacant, Spain 2 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain Full list of author information is available at the end of the article Page 2 of 17 Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 members are involved in synaptic plasticity regulation and neuronal migration (extensively reviewed in [1–6]). LDL receptors are related to Alzheimer’s disease (AD) pathogenesis as receptors of apolipoprotein E (apoE) [7], being the APOE4 variant the largest known genetic risk factor for late-onset sporadic AD [8, 9]. Additionally, several members of the LDL receptor family are able to modulate the amyloid precursor (APP) proteolytic processing, either by regulation of the generation of the β-amyloid peptide (Aβ) or through Aβ clearance [10–13]. An important member of the LDL receptor family, ApoER2/LRP8, can exert a modulatory effect in transcriptional expression. ApoER2 interaction with its ligand, the reelin protein, drives to a sequential proteolytic processing, resulting in the cleavage of the receptor by α-secretase, which generates a membrane-tethered C-terminal fragment (ApoER2-CTF), followed by the cleavage by γ-secretase. The action of γ-secretase generates an intracellular domain fragment (ApoER2-ICD) capable of decreasing the expression of reelin mRNA [14, 15]. Using the same brain extracts as in [14], we found later that the generation of ApoER2-CTF appeared lower and, accordingly, reelin expression resulted higher with respect to those in control brain extracts [16]. In this study, we have further explored the modulatory transcriptional activity of ApoER2/reelin signaling, and we have observed that this pathway can modulate the expression of the LDL-related protein 3 (LRP3). LRP3 is probably the most unknown member of a new subfamily of LDL receptors [17], whose precise role in the central nervous system is still undetermined. We have estimated LRP3 expression in the frontal cortex of middle-aged (MA) individuals and in cases with Alzheimer’s disease (AD)-related pathology, and after overexpression in CHO cells. We have demonstrated that LRP3 is able to modulate APP expression. Material andmethods 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 postmortem 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 Page 3 of 17 Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 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 Table 1 Human samples Age (y) Gender PM (h) SP ApoE MA NFT 0 46 f 9.5 0 ɛ2/ɛ3 46 m 15 ɛ3/ɛ4 47 m 5 ɛ3/ɛ3 49 m 7.5 ɛ3/ɛ3 50 m 17 ɛ3/ɛ3 52 m 5 ɛ3/ɛ3 52 f 6 ɛ4/ɛ4 53 m 7.5 ɛ3/ɛ3 56 m 4 ɛ2/ɛ3 59 m 6,5 ɛ3/ɛ3 60 f 11.5 ɛ3/ɛ3 AD NFT Braak I 53 m 6.25 A ɛ3/ɛ4 64 m 8.5 0 ɛ3/ɛ3 67 m 14.5 0 ɛ3/ɛ3 68 m 11 0 ɛ2/ɛ3 Braak II 57 m 4.5 0 ɛ3/ɛ4 60 f 9.5 A ɛ3/ɛ3 65 m 16.5 0 ɛ3/ɛ3 67 m 7.25 0 ɛ3/ɛ4 69 m 3.5 A ɛ3/ɛ4 72 m 6.25 A ɛ3/ɛ4 74 m 5.5 A ɛ2/ɛ3 78 m 16 0 ɛ3/ɛ3 78 m 10.75 B ɛ3/ɛ4 86 m 5.5 A ɛ2/ɛ3 Braak III 68 f 4.5 A ɛ3/ɛ3 71 m 7.5 0 ɛ2/ɛ3 73 m 4 0 ɛ3/ɛ3 76 f 4 B ɛ3/ɛ3 77 m 13.5 C ɛ3/ɛ4 77 m 5.5 A ɛ3/ɛ3 79 f 3.5 B ɛ3/ɛ3 82 f 5 A ɛ3/ɛ3 90 f 4 B ɛ3/ɛ3 Braak IV 79 m 5 A ɛ4/ɛ4 81 f 5 C ɛ3/ɛ3 85 m 14 B ɛ3/ɛ4 89 m 3.5 B ɛ3/ɛ4 99 f 5 B ɛ3/ɛ3 Braak V 72 m 2.75 C ɛ3/ɛ4 73 m 4.5 B ɛ3/ɛ4 74 f 9 A ɛ3/ɛ4 75 m 11.5 B ɛ3/ɛ4 77 m 16 C ɛ3/ɛ3 78 m 17 0 ɛ3/ɛ3 81 f 5.5 C ɛ3/ɛ4 87 m 7 C ɛ3/ɛ3 93 m 3 C ɛ3/ɛ3 Table 1 (continued) Age (y) Gender PM (h) SP ApoE Braak VI 56 f 7 C ɛ3/ɛ3 67 f 8 C ɛ3/ɛ4 86 f 20.5 C ɛ3/ɛ3 Middle‑aged (MA) cases and cases with AD‑related pathology (AD). Subjects were categorized according to the Braak stage of neurofibrillary tangle (NFT I–VI) and senile plaque staging (0–C) [18, 19]. Age (y years), gender (m male, f female), post‑mortem (PM, h hours), SP senile plaques, APOE (APOE alleles, ɛ2, ɛ3, and ɛ4) Page 4 of 17 Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 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. bioar ray. 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 MicroarrayBased 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: AAC CAG TGA CCA TCC AGA AC; reverse: ACT TGT CAG GAA CGA GAA GG) and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, forward: AGA AGG TGG TGA AGC AGG CAT; reverse: AGG TCC ACC ACT CTG TTG CTGT) 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 0.2μm pores and concentrated with an Amicon Ultra 100kDa 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, SigmaAldrich, 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% Page 5 of 17 Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 paraformaldehyde and 0.1 M EGTA for 10 min. To stain the plasma membrane, cells were incubated with WGAFITC (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, paraffinembedded, 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. Results ApoER2 overexpression increases theexpression ofLRP3 SH-SY5Y cells were transfected with full-length ApoER2, and after 48 h, a microarray was performed. Among the genes affected, we focused on the analysis of LDL receptors and apolipoprotein-related genes (Table 2). The receptors LRP3 and LDLR appeared significantly upregulated, both of which are members of the LDL receptor family. Upregulation of LRP3 was confirmed by qRT-PCR, with a significant increase in mRNA LRP3 level compared to its expression in non-transfected cells. However, increments in LDLR mRNA expression were not significant when assessed by qRT-PCR (Fig.1a). Although SH-SY5Y cells secrete reelin to the media and it can act in a paracrine mode, recombinant reelin was employed to treat overexpressing-ApoER2 cells to potentiate the ApoER2 signaling. This treatment induced ApoER2 cleavage and, consequently, reduced the amount Table 2 Expression of genes upregulated by full‑length ApoER2 overexpression Genes associated with lipid binding and transport, and cholesterol metabolism, whose transcripts were upregulated in ApoER2 overexpressing SH‑SY5Y cells compared with control cells transfected with an empty vector. The expression of the genes was analyzed on DNA microarrays. The fold change (logFC) in gene expression between samples and controls, as well the adj p (p value adjusted for multiple testing) is indicated Symbol Gene name Genomic location Function logFC adj p LRP3 Low‑density lipoprotein receptor‑ related protein 3 19q13.11 Internalization of lipophilic molecules and/or signal transduction Precise role is unclear 0.48 0.047 LDLR Low‑density lipoprotein receptor 19p13.2 Mediates endocytosis of cholesterol‑rich LDL 0.43 0.018 APOL1 apolipoprotein L, 1 22q12.3 Minor apoprotein component of HDL 1.28 0.003 INSIG1 Insulin‑induced gene 1 7q36.3 Regulation of cholesterol cell concentration 0.72 0.001 DHCR24 24‑Dehydrocholesterol reductase 1p32.3 Cholesterol metabolic process 0.35 0.008 MVK Mevalonate kinase 12q24.11 Cholesterol metabolic process 0.33 0.019 Page 6 of 17 Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 Fig. 1 ApoER2/reelin signaling upregulates LRP3 expression. a qRT‑PCR analysis showing expression of LRP3 mRNA and LDLR mRNA after transfection with GFP cDNA (control) and full‑length ApoER2 cDNA (fApoER2) in SH‑SYSH cells. 18S was used as an internal control for mRNA expression (n = 10–12 for each condition, p < 0.001 for control versus fApoER2; t‑test with Welch’s correction). Note that the X axis begins at 50%. b Quantification and western blot showing the expression of full‑length ApoER2, ApoER2‑CTF, and LRP3 proteins after fApoER2 transfection and reelin (12 μg/ml) treatment for 24 h in SH‑SY5Y cells. Tubulin was used as an internal control (n = 9 for each condition, **p < 0.001 for expression of fApoER2, t‑test with Welch’s correction, and ApoER2‑CTF, t‑test; *p < 0.05 for expression of LRP3, t‑test). c Quantification and western blot showing the expression of LRP3 protein after reelin (12 μg/ml) treatment for 24 h or vehicle (Hanks’s media) in neuro‑differentiated SH‑SY5Y cells with retinoic acid (n = 9 for each condition, *p <0.05 t‑test) Page 7 of 17 Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 of full-length ApoER2 and increased the generation of the ApoER2-CTF. Importantly, reelin treatment induced an increment of LRP3 protein levels (Fig. 1b). In RA neuro-differentiated SH-SY5Y cells, reelin treatment was also able to induce an increase in LRP3 protein levels compared to non-stimulated cells (Fig.1c). Expression ofApoER2‑ICD upregulates LRP3 expression We considered the possibility that increments of LRP3 expression were induced by ApoER2-ICD, a fragment with transcriptional regulatory activity [14], generated by the proteolytic cleavage of ApoER2-CTF. This small fragment was observed in ApoER2-overexpressing cells after treatment with reelin (Fig.2a). Thus, we overexpressed a chimeric ApoER2-ICD (amino acid residues 728–842) and measured LRP3 expression. LRP3 mRNA expression and protein levels increased significantly with respect to non-transfected cells (Fig.2b–d), while LDLR mRNA levels were not significantly affected by ApoER2-ICD (Fig.2e). Expression levels ofLRP3 inAβ42‑treated cells On the contrary to the upregulation of LRP3 mRNA and protein that we observed after overexpression of full-length ApoER2 or ApoER2-ICD, we expected to find less LRP3 expression in Aβ42-treated cells, due to the fact that Aβ treatment reduces the generation of ApoER2-CTF [16]. In agreement with this view, we found that treatment of neuro-differentiated SH-SY5Y cells with 1 μM and 5μM Aβ42 decreased the LRP3 protein levels, but 500 nM did not have the same effect, in comparison to scrambled peptide treatment (control, Fig.3a). Five micromolar Aβ42 also reduced LRP3 mRNA expression (Fig.3b). Expression levels ofLRP3 inAD brain Next, we examined LRP3 levels in human frontal cortex extracts. Considering all cases with AD-related pathology, LRP3 mRNA expression was lower with respect to MA subjects (p = 0.02; t-test) (Fig.4a). However, when cases with AD-related pathology were categorized by Braak NFT stages, the reduction was significant only at Braak stages NFT I–II (p = 0.03; t-test), while NFT III–IV or NFT V–VI displayed the same trend but failed to reach statistical significance (p = 0.10; p = 0.15, respectively, t-test). No significant modifications were found between Braak stages NFT I–II and NFT III–IV or NFT V–VI (p = 0.56; p = 0.65, respectively, t-test; Fig.4b). Despite the difference in age between MA and AD-related pathology cases, age did not correlate with LRP3 mRNA in MA (n = 11; R = 0.058, p = 0.87) or AD-related pathology individuals (n = 40; R = 0.067; p = 0.68). Gender did not contribute to differences in LRP3 mRNA expression either. The comparison between Fig. 2 ApoER2‑ICD increases LRP3 expression. a Representative western blot showing the expression of ApoER2‑ICD after transfection with full‑length ApoER2 cDNA (ApoER2) and reelin treatment (12μg/ml, ApoER2 + reelin) for 24 h in SH‑SY5Y cells. For comparison, the expression of the ApoER2‑ICD construct (ApoER2‑ICD) is also shown. b Western blot and c quantification of LRP3 protein expression after ApoER2‑ICD transfection in SH‑SY5Y cells. Tubulin was used as an internal control (n = 6 for each condition, *p < 0.001, t‑test). d qRT‑PCR analysis showing the expression of LRP3 mRNA (n = 7 for each condition, *p < 0.05, t‑test) and e LDLR mRNA (n = 10 for each condition) after transfection with GFP cDNA (control) and ApoER2‑ICD cDNA in SH‑SY5Y cells. Note that the X axis in d begins at 50%. 18S was used as an internal control for mRNA expression Page 8 of 17 Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 Fig. 3 Aβ42 reduces LRP3 expression. a Quantification and western blot showing the expression of LRP3 proteins in neuro‑differentiated SH‑SY5Y cells treated with 5 μM Aβ42 or scrambled Aβ42 (control). Tubulin was used as an internal control (n = 9 for each condition, *p < 0.05, t‑test). b qRT‑PCR analysis showing expression of LRP3 mRNA in neuro‑differentiated SH‑SY5Y cells treated with 500 nM, 1μM, 5 μM Aβ42, or scrambled Aβ42 (control). 18S was used as an internal control for mRNA expression (n = 8 for each condition, p < 0.05; t‑test) Fig. 4 Low levels of LRP3 in AD frontal cortex. a qRT‑PCR analysis showing expression of LRP3 mRNA in brain extracts from MA and Alzheimer’s disease‑related (AD‑r) subjects, and b categorized by Braak NFT stages (I–II, III–IV, and V–VI). 18S was used as an internal control for mRNA expression (n = 11 for MA, n = 12–14 for each AD‑r Braak stage, *p < 0.05, t‑test for MA v AD‑r, t‑test with Welch’s correction for MA v AD‑r I–II). c Western blots showing different LRP3 immunoreactivities in human cortex extracts. Two bands were observed using an anti‑C‑terminal LRP3, but a single band was observed when an anti‑N‑terminal LRP3 was used, all between 70 and 100kDa. One of the bands immunoreacted to both antibodies, likely representing the full‑length receptor. Accordingly, the overlapping band (*) was selected for quantification. d Western blot using an anti‑C‑terminal LRP3 in brain extracts from MA and AD‑r subjects, categorized by Braak’s stages (NFT I–II, NFT III–IV, and NFT V–VI) and quantification of the lower band (marked with a *). Tubulin was used as an internal control (n = 11 for MA, n = 10–11 for each NFT Braak’s stage, *p < 0.05, Mann‑Whitney test). e qRT‑PCR analysis showing expression of LDLR mRNA in brain extracts from MA and AD‑r subjects, categorized by Braak’s stages. 18s was used as an internal control for mRNA expression (n = 9 for MA, n = 10 for each Braak’s stage) Page 9 of 17 Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 females and males from MA and from AD-related pathology groups was not statistically significative (p = 0.13, one-way ANOVA). When female values were subtracted from both groups, LRP3 mRNA expression in males was still different between MA and AD-related pathology overall (p = 0.042, t-test). However, the difference observed in Braak stages I–II failed to maintain statistical significance, probably due to the smaller sample size (p = 0.060, t-test). Braak stages III–IV and V–VI remained without differences in males compared to MA males (p = 0.20 and p = 0.22, respectively, t-test). The APOE genotype did not account for LRP3 mRNA expression either (p = 0.47 ɛ4 carriers v non-ɛ4 carrier AD-related cases). To evaluate LRP3 protein levels in the cortex from MA and cases with AD-related pathology, membraneenriched fractions were isolated from brain samples. Due to the lack of reports about LRP3 in the brain, two antibodies were tested to corroborate the identity of LRP3 immunoreactive bands (Fig. 4c). We found that LRP3 expression levels were lower at Braak stages I–II compared to those in MA individuals (p = 0.048, t-test, Fig.4d). No further differences were seen at stages III–IV and V–VI when compared with MA (p = 0.11 and p = 0.12, respectively, t-test) and compared with Braak NFT stages I–II (p = 0.84 and p = 0.26 respectively, t-test). The estimated expression of LDLR mRNA was not significantly different between MA individuals and AD-related pathology subjects when the extracts were compared overall (p = 0.73 Mann-Whitney) or when compared discriminating Braak stages (p = 0.73 one-way ANOVA; Fig.4e). LRP3 interacts withapoE andAPP, butnotwith reelin inthehuman brain Double-labeling immunofluorescence and confocal resolution showed that the LRP3 antibody recognized small granules localized in the cytoplasm and proximal dendrites of all neurons, and around the nucleus of glial cells in the hippocampus and frontal cortex. ApoER2 antibody also showed small granules in the cytoplasm of neurons and small glial cells. The immunostaining was variable in the MA group and in cases with NFT pathology with marked individual disparities, probably due to the vulnerability of the protein to the pre-mortem status and post-mortem delay (Fig. 5a). This individual variability did not permit any attempt to quantify inter-group immunostaining densitometry. We also evaluated, by means of immunoprecipitation assays, whether reelin acts as a ligand for LRP3, as it does for ApoER2, in frontal cortex extracts from MA and AD-related pathology cases. Reelin was not co-immunoprecipitated from any brain extracts. We next assessed whether LRP3 interacts with apoE and APP, in the same way as many members of the LDL receptor family do. After immunoprecipitation, both proteins were coimmunoprecipitated with LRP3 in MA and cases with AD-related pathology (Fig.5b). LRP3 modulates APP expression levels We tested whether LRP3 was able to influence APP processing and Aβ generation in a similar manner to other members of the LDL receptor family. In order to do so, we overexpressed LRP3 in CHO-PS70 cells, a cell line that expresses the wild-type APP770 isoform. LRP3 was located at discrete areas of the soma and in the plasma membrane of CHO-PS70 cells (Fig.6a). Moreover, LRP3 and APP co-immunoprecipitated in these cells (Fig.6b). Overexpression of LRP3 did not affect APP mRNA levels (Fig.6c), but it drastically reduced full-length APP levels, as well as APP-CTF in cell extracts (Fig.6d). In the supernatant, the levels of sAPPα, sAPPβ, and soluble Aβ decreased in transfected CHO-PS70 cells compared to mock-transfected cells (Fig.6e). Interestingly, when lysosomal function was impaired by chloroquine, full-length APP and sAPPα levels increased in a significant manner with regard to non-treated cells (p = 0.0044; p = 0.031, respectively, t-test; Fig.7). sAPPβ levels showed a tendency to be higher than non-treated cells (p = 0.065). To determine in more detail whether LRP3 is involved in APP degradation by lysosomes, we performed a differential centrifugation of CHO-PS70 cell homogenates. Two different fractions were obtained: a cytosol and plasma membrane-containing fraction, and an intracellular membrane-containing fraction. In CHO cells overexpressing LRP3, full-length APP levels were lower in both fractions, but APP-CTF levels were lower only in the intracellular membrane-containing fractions compared to those in CHO controls (Fig.8a). Treatment with chloroquine did not affect APP levels in CHO cell controls in any fraction (Fig.8b). In CHO cells overexpressing LRP3, full-length APP and APP-CTF levels increased in the cytosol and plasma membrane-containing fractions after chloroquine treatment. This could indicate that chloroquine is affecting LRP3 capacity of inducing APP endocytosis from the plasma membrane as observed in Fig.8a. However, only APP-CTF levels were higher than those in CHO controls in the intracellular membrane-containing fractions (Fig.8c). This could indicate an accumulation of APP-CTF in vesicles such as endosomes or autophagosomes, whose fusion with lysosomes is inhibited by chloroquine. Discussion Our results suggest that reelin signaling, through the cleavage of its receptor ApoER2, can ultimately influence the expression of other liporeceptors, such as LRP3. Page 16 of 17 Cuchillo‑Ibañezetal. Alz Res Therapy (2021) 13:181 36. 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