The β-amyloid peptide compromises Reelin signaling in Alzheimer’s disease

Reelin is a signaling protein that plays a crucial role in synaptic function, which expression is influenced by β-amyloid (Aβ). We show that Reelin and Aβ oligomers co-immunoprecipitated in human brain extracts and were present in the same size-exclusion chromatography fractions. Aβ treatment of cells led to increase expression of Reelin, but secreted Reelin results trapped together with Aβ aggregates. In frontal cortex extracts an increase in Reelin mRNA, and in soluble and insoluble (guanidine-extractable) Reelin protein, was associated with late Braak stages of Alzheimer’s disease (AD), while expression of its receptor, ApoER2, did not change. However, Reelin-dependent induction of Dab1 phosphorylation appeared reduced in AD. In cells, Aβ reduced the capacity of Reelin to induce internalization of biotinylated ApoER2 and ApoER2 processing. Soluble proteolytic fragments of ApoER2 generated after Reelin binding can be detected in cerebrospinal fluid (CSF). Quantification of these soluble fragments in CSF could be a tool to evaluate the efficiency of Reelin signaling in the brain. These CSF-ApoER2 fragments correlated with Reelin levels only in control subjects, not in AD, where these fragments diminished. We conclude that while Reelin expression is enhanced in the Alzheimer’s brain, the interaction of Reelin with Aβ hinders its biological activity.

. Reelin and Aβ interact in human brain tissue. Immunoprecipitation of human frontal cortex extracts, non-disease control (ND) and AD with (a) anti-Reelin (G10+ CR50), or (b) anti-Aβ (4G8) antibodies (n = 3; T, total lysate). The immunoprecipitated proteins (IP) were probed with the 142 antibody against Reelin and the 6E10 antibody against Aβ . The Reelin antibody detects the 420 kDa full-length and the 310 and 180-kDa fragments. Extracts incubated with beads in the absence of antibody were negative controls (IPc). (c) Reelin blot from cortex extracts fractioned by size-exclusion. (d) Reelin-rich fractions were also pooled together and probed with the 4G8 antibody against Aβ , and analyzed for comparison with fractions containing small Aβ oligomers. Note that Reelin co-immunoprecipitates and interacts mostly with oligomeric Aβ species. One AD case is shown but similar elution profiles were obtained for ND cases.
Scientific RepoRts | 6:31646 | DOI: 10.1038/srep31646 We also tested the effect of Aβ 42 on differentiated SH-SY5Y cells that secrete Reelin. Remarkably, the mRNA Reelin content was increased in cellular extracts treated with the peptide (Fig. 2a). Accordingly, exposing these cells to Aβ 42 augmented the cellular Reelin protein levels relative to untreated cells, while reduced amount of secreted Reelin was detectable into the culture media (Fig. 2b). A previous study indicated that increased Reelin levels in cell treatment with Aβ 42 did not result from altered secretory pathway 17 . When the material pelleted by centrifugation of the culture media was examined, we found a large amount of Reelin together with Aβ oligomers almost exclusively in Aβ -treated cells (Fig. 2c), indicating that a notable amount of secreted Reelin could be associated with the Aβ fibers formed during the 4 day exposure to the peptide. Since Aβ 42 exposure induced a degree of death cell (22 ± 3%, MTT reduction; p < 0.001), some of the Reelin recovered from the media may originate from these dead cells.
Reelin expression increases in the brain of AD patients. Reelin expression in the frontal cortex of AD and ND was evaluated by qRT-PCR. When AD cases were classified with respect to their Braak and Braak stage, we found a two-fold increase in relative Reelin mRNA expression in extracts from late AD Braak stages (V to VI; p = 0.03) with respect to ND (Fig. 3a). In contrast, no differences were observed at Braak stages I-II to V. We also determined the Reelin expression in the hippocampus by qRT-PCR at Braak stages IV to VI (the only tissue available from our collection) and again, Reelin mRNA was expressed more strongly in the AD hippocampus at these stages that in the ND tissue (p = 0.02; Fig. 3b).  . Increased Reelin expression in the brain at advanced Braak stages of AD. AD subjects were categorized as early (I-II to IV) or advanced Braak stages (V to VI). (a) Relative Reelin mRNA expression analyzed by qRT-PCR in frontal cortex samples from ND (n = 11) and AD subjects (stages I to IV, n = 7; stages V to VI, n = 10). (b) Reelin mRNA was also analyzed in the hippocampus of ND controls (ND; n = 5) and AD samples corresponding to Braak stages IV to VI (n = 9). The values were calculated from relative standard curves and normalized to GAPDH from the same cDNA preparation, confirming the specificity of the PCR products from dissociation curves. The data represent the means ± SEM. * p < 0.05 using Student's t-test. We also compared the levels of soluble full-length Reelin in Western blots of AD and ND brain extracts. We found a significant increase in full-length Reelin in extracts from advanced stages of AD (stages V to VI, 85% increase; p = 0.02) with respect to the ND extracts (Fig. 4a). Similarly, an increase in the major 180 kDa Reelin fragment was also evident at these stages with respect to ND (186% increase; p = 0.003). By contrast, no significant differences were found between extracts from earlier Braak stages (I-II to IV) and ND extracts.
Since previous cell culture experiments suggested that Reelin could be sequestered into Aβ fibers, we also analyzed the levels of Reelin in amyloid pellets. Brain tissue pellets were re-suspended and solubilized with GuHCl to extract the insoluble amyloid from AD brains 20 , and when the GuHCl extractable full-length Reelin was quantified ( Fig. 4b) the same tendency as that seen for soluble Reelin was observed. Thus, there was significantly more full-length Reelin in AD samples from Braak stages V to VI (114% increase; p = 0.001) than in ND extracts but not from earlier Braak stages (I-II to IV).
Handling could influence the stability and measurement of Reelin, particularly its denaturation during sample preparation for electrophoresis 16,21 . Since Reelin isolated from the AD brain exhibits a different glycosylation pattern in comparison to Reelin from ND extracts 16,17 , sample handling could affect different Reelin glycoforms to a different extent. In fact, a 5 min boiling of the sample prior to SDS-PAGE apparently diminished the Reelin in the AD cortex by up to 70% compared to that detected after 3 min at 98 °C, whereas the Reelin in the cortex from ND cases was less strongly affected by 5 min boiling, diminishing it only for ~25% with respect to 3 min denaturation. These data suggested that Reelin from AD extracts is particularly sensitive to temperature, and that the differences between the Reelin in AD and ND extracts could be influenced by temperature (see Supplementary Fig. 1). In this study, a 3 min denaturation at 98 °C was used to study Reelin in Western blots.
In order to confirm the results from the Western blots, brain Reelin was also quantified using an ELISA specific for human Reelin (USCN, Life Science Inc) that does not involve sample denaturation by heating. To our knowledge, this kit has not been validated previously and thus, we first confirmed that this ELISA could detect full-length and N-terminal fragments of brain Reelin by analyzing the unbound fraction in Western blots. When fresh aliquots of AD and ND brain extracts were analyzed Reelin protein was increased ~97% (p = 0,043) in the entire AD group (0.18 ± 0.03 ng/ml) with respect to that in the ND subjects (0.09 ± 0.01 ng/ml).

ApoER2 expression does not change but Dab1 is less phosphorylated in AD brain. Levels of
ApoER2 were analyzed in ND and AD brain extracts. There were no significant differences in the amount of full-length ApoER2 protein, quantified by Western blots (Fig. 5a), neither in the ApoER2 mRNA quantified by qRT-PCR (Fig. 5b) between AD, for any Braak stage, and ND subjects.

Figure 4. An increase in Reelin protein in the frontal cortex at advanced Braak stages of AD.
Frontal cortex samples from ND (n = 11) and AD subjects (n= 17) were homogenized in detergents diluted in Trissaline buffer (soluble Reelin, (a) and the pellets recovered were re-extracted in Guanidine-HCl (GuHCl Reelin, (b). Western blots were probed with the 142 antibody (α -tubulin served as a loading control). AD subjects were categorized as early (I-II to IV; n = 7) or advanced Braak stages (V to VI; n = 10). The data represent the means ± SEM and were normalized with respect to the ND values. The data represent the means ± SEM. * p < 0.05, * * p < 0.01, * * * p < 0.001, using Student's t test.
Scientific RepoRts | 6:31646 | DOI: 10.1038/srep31646 Phosphorylation of the downstream protein Dab1 was also examined. Quantitative analyses showed a decrease (30%; p = 0,01) in tyrosine phosphorylation of Dab1 in the entire AD group with respect to that in the ND group (Fig. 5c). A different combination of P-Dab1/Dab1 antibodies served to ensure the identity of the immunoreactive bands and confirm the decrease (36%; p = 0,004) in tyrosine phosphorylation of Dab1 ( Supplementary Fig. 2).
Aβ impairs Reelin signaling. The effect of Aβ 42 on Reelin signaling was tested by analyzing the fate of ApoER2 in protein-surface biotinylated SH-SY5Y cells. Cells overexpressing full-length ApoER2-Cherry were stimulated with recombinant Reelin (~10 nM), Aβ 42 or with Reelin (~10 nM) plus Aβ 42 previously incubated together. After 15 min at 37 °C, the surface biotinylated proteins were pulled down and probed in Western blots with an antibody against ApoER2 (Fig. 6a). Reelin reduced the presence of ApoER2 at the cell membrane with respect to the controls, but when cells were treated with Reelin and 2 μ M Aβ 42, plasmatic membrane ApoER2 exposition increased noticeably ( p = 0.04).
Reelin binding induces the clustering and the proteolytic processing of ApoER2, generating a soluble extracellular fragment 22,23 , that can be recognized by the 186 antibody raised against the entire ligand binding domain of the receptor 24 . The generation of this extracellular fragment could be observed in SH-SY5Y cells over-expressing ApoER2 after treatment with Reelin, which induces the appearance of a ~70 kDa ApoER2 fragment in the culture medium (Fig. 6b). In contrast, a lower amount of this soluble fragment was measured in the medium of cells treated with Reelin plus Aβ 42 (Fig. 6c). (a) Quantification of ApoER2 (~130 kDa + ~170 kDa) in the ND (n = 10) and AD cortex (n = 12) in Western blots probed with a C-terminal ApoER2 antibody and α -tubulin as loading control. (b) Relative ApoER2 mRNA expression analyzed by qRT-PCR in ND (n = 8) and AD (n = 11) frontal cortex, calculated from standard curves and normalized to GAPDH. Data represent the mean ± SEM. None of the comparisons resulted in significant differences, considering the entire AD group or the advanced Braak stages. (c) Western blots of frontal cortex extracts from ND (n = 12) and AD (n = 15) revealed by fluorescence simultaneously for an anti-Dab1 antibody and an anti-phosphotyrosine (P-Tyr), and α -tubulin as loading control. Data represent the means ± SEM and were normalized with respect to the ND values. * p < 0.005 using a Student's t test.
Previously we demonstrated that Reelin obtained from SH-SY5Y cells treated with Aβ 42 fails to reduce tau phosphorylation 19 . Here we tested if Aβ directly impairs the effect of Reelin in the modulation of tau phosphorylation. Neurons treated with Reelin in presence of Aβ 42 fail to induce phosphorylation of Dab1 and to reduce tau phosphorylation (see Supplementary Fig. 3). These results confirm that Aβ compromises the biological role of Reelin modulating tau phosphorylation.
Evidences for a Reelin signaling impairment in AD. Following our interest to study the malfunctioning of Reelin signaling in the AD brain, we postulated that quantifying the soluble ApoER2 fragments in the CSF would reflect the efficiency of Reelin signaling, given that we have not found traces of full-length ApoER2 in cerebrospinal fluid (CSF). Indeed, we found a reduction of the ~70 band detected by the 186 antibody in the CSF from a heterozygous Reelin mutant sheep 25 (Fig. 7a). Likewise, the 186 antibody detected similar ApoER2 fragments in human CSF from ND cases (Fig. 7b), with the most abundant 70 kDa ApoER2 fragment positively correlated with the full-length Reelin levels (r = 0.85, p < 0.001; Fig. 7c).
The soluble ApoER2 fragment was measured in the CSF from AD and age-matched ND subjects. The CSF samples were selected on the basis of a similar Reelin content (Fig. 8a); Reelin from AD CSF also displayed the characteristic altered glycosylation pattern of Reelin from ND samples 17 . In the CSF from AD subjects there was a significant decrease (~54%) in the 70 kDa soluble ApoER2 fragment, with respect to that from ND subjects (Fig. 8b). Moreover, the ApoER2 fragments were not correlated with the amount of Reelin in AD samples, suggesting a disparity between Reelin and ApoER2 in AD (Fig. 8c).
Altogether, these results indicate that the Reelin present in the AD brain is targeted by Aβ . The association between these proteins hinders the processing of ApoER2 and probably Reelin signaling in AD.

Discussion
A "functional" link between Reelin and Aβ has previously been demonstrated, whereby Reelin delays Aβ fibril formation in vitro 13 and it protects against toxicity in vivo 13,26 . In anatomical studies, Reelin has been found associated with Aβ in transgenic mice model of AD and in aged wild-type mice 12,27,28 . However, the interaction between Reelin and Aβ in human AD brains had not been explored so far. In this study, we present evidence that soluble Reelin interacts with Aβ in ND and AD human brain homogenates. This interaction elicits an increase in Reelin expression in cells, but decreases the internalization of ApoER2 from the cytoplasmic membrane; thus Aβ hinders Reelin biological activity and ultimately could influence pathological progression of Alzheimer's disease by impairing Reelin signaling.
In frontal cortex tissue from advanced stage AD patients (Braak stage V to VI), we found a significant increase in soluble and GuHCl extractable Reelin. These advanced Braak stages correspond to the phase of neocortical lesion expansion into high order association areas, such as the frontal, parietal and occipital neocortex 29 . Increased Reelin expression was also demonstrated by qRT-PCR in the frontal cortex at late Braak stages, as confirmed in the hippocampus, another brain area affected in AD. An up-regulation of Reelin transcripts in the frontal cortex of AD patients has been proposed previously 16,17,30 . Moreover, large increases in Reelin protein and mRNA have also been described in the brain of individuals with Down syndrome, where APP is overexpressed 17 . Thus, the dynamics of the Reelin and Aβ interaction, and the associated changes provoked in Reelin expression, appear to be related to disease progression, a phenomenon that merits further investigations. We presume that oligomeric Aβ is the specie that triggers the increase in Reelin expression; however, under the conditions of our cellular experiments, we cannot differentiate if this effect is due to oligomeric Aβ or fibers. Anyhow, Aβ fibers might have also a role in compromising Reelin activity since Reelin results trapped into Aβ aggregates.
There are currently some discrepancies regarding the levels of Reelin in the brain. In some studies Reelin depletion has been described in AD brains 14,15 , whereas an increase in Reelin has also been observed 16,17,18 . These differences could be due to the reduced sample size used in some studies, while factors related to the handling of samples also emerge as a potentially important cause to influence the evaluation of Reelin protein (discussed in ref. 31). Here, we demonstrated that heating affects the detection of Reelin in protein extracted from AD brains, which displays differences in glycosylation. It is well known that changes in glycosylation can influence protein stability and limit a protein's half-life in circulation 32,33 . However, the ELISA assay used here, which allows protein content to be determined without heating the samples, confirmed that soluble Reelin levels are higher in the AD cortex.
A relevant question that still remains is whether Reelin is biologically active in the AD brain and consequently, whether the integrity of Reelin signaling is retained. Previous studies described that the levels of the intracellular adapter Dab1 mRNA are upregulated 34 or unaffected 18 in frontal cortex of AD patients, although the study of Dab1 phosphorylation has not been addressed yet. Our data indicate an abnormal Reelin signaling in the brain of AD patients, since levels of phosphorylated Dab1 were decreased respect to total Dab1. Moreover, we demonstrate that Aβ can directly interfere with the Reelin-dependent internalization of its receptor, ApoER2. ApoER2  appears to be the dominant Reelin receptor in the human forebrain, participating in the modulation of synaptic plasticity and memory formation 35,36 . We examined the presence of a proteolytic ApoER2 fragment in the CSF, generated by cleavage of the receptor after Reelin binding. We quantified this soluble ApoER2 fragment in CSF as a read-out of Reelin signaling, which appears to be a better indicator than Reelin fragments. Although Reelin processing is derived to some extent from its interaction with the receptor 37-39 , fragments of Reelin can also be generated through the activity of extracellular matrix metalloproteinases [40][41][42][43] , independent of ApoER2, ruling out the quantification of Reelin fragments as a suitable read-out of its signaling function. In CSF from ND subjects, the levels of ApoER2 fragments correlate with that of full-length Reelin, while in AD subjects this correlation does not exist and the levels of the soluble ApoER2 fragment decreases, even though ApoER2 expression did not change. Therefore, while there is more Reelin in the AD brain, less soluble ApoER2 fragments are found in the CSF. As such, quantifying the circulating ApoER2 fragments may be useful to assess the effects on Reelin signaling in other neuropsychiatric disorders where the expression of Reelin is altered 44 . In this context, to assay levels of ApoER2 transcripts in brain of AD subjects we designed primers common for all splicing variants. Recent finding indicate that the balance of ApoER2 splicing variant is deregulated in brain from AD patients and in a transgenic mouse model of AD 45 . Indeed, differential splicing and glycosylation of ApoER2 regulates its role in synaptic function and memory 46 . Further studies should clarify the role of deregulated ApoER2 splicing in AD.
Cleavage of ApoER2 occurs through sequential α -and γ -secretase processing, resulting in the release of a soluble intracellular domain (ICD) 5,23 . We recently reported that this ApoER2-ICD down-regulates RELN promoter activity and consequently Reelin expression 47 . While there is an increase in Reelin in the AD cortex, this Reelin is glycosylated distinctly to that in the ND cortex 17 . The altered Reelin glycosylation induced by Aβ appears to impair its efficient binding to ApoER2, dampening the down-regulation of tau phosphorylation via the GSK3β kinase 19 . Thus, Aβ could establish a vicious circle in the pathological condition, whereby a less-functional Reelin would generate fewer ApoER2-ICD fragments that would in turn increase Reelin transcription, as occurs in the AD brain. Therefore, the effect of Aβ on Reelin in the AD brain might induce chronic signaling failure, which would consequently affect synaptic neurotransmission, plasticity and memory.
Finally, the most robust evidence that links impaired Reelin-ApoER2 signaling with AD neurodegeneration might be the increase in tau phosphorylation. Indeed, tau phosphorylation and fibrillary tangles are more closely associated with the severity of memory loss in humans than Aβ [48][49][50] . Perhaps the influence of Aβ in the impaired Reelin brain function reveals a cross-talk between disturbed tau phosphorylation and β -amyloid. Previous data demonstrated that Reelin forms induced by β -amyloid are less capable of down-regulating tau phosphorylation via Dab1 and GSK3β 19 . Our data associates Aβ and tau phosphorylation dysregulation through Reelin and raises the possibility that Reelin directly contributes to the progress of AD pathology. The mechanism of mutual influencing and the role of Reelin deserve attention.

Methods
Collection of human brain and CSF samples. This study was approved by the ethic committee of Universidad Miguel Hernández de Elche, Spain, and it was carried out in accordance with the Helsinki Declaration. Brain samples (frontal cortex and hippocampus) were obtained from the UIPA neurological tissue bank (Unidad de Investigación Proyecto Alzheimer; Madrid), in which sporadic AD cases [n = 17 (9 female/8 male); 83 ± 1 years] were categorized according to the Braak and Braak stage 29 . Samples from ND individuals (n = 11 (4 female/7 male); 63 ± 3 years) corresponded to individuals with no clinical dementia and no evidence of a brain pathology. The mean post-mortem interval of the tissue was 6 h in all cases, with no significant difference between the groups. CSF samples from probable AD cases (n = 10; 77 ± 2 years) and ND controls (n = 8; 72 ± 3 years) were collected at the "Hospital Clínico San Carlos", Madrid, Spain. All experiments were carried out in accordance with the Helsinki Declaration guidelines and regulations. All experimental protocols were approved by ethic committee of Hospital Clínico San Carlos and Universidad Miguel Hernández de Elche. An informed consent was obtained from all subjects. All AD patients fulfilled the NINCDS-ADRDA criteria for "probable" AD 51 .
CSF samples were also collected from two 5-year-old sheep heterozygous for a naturally-occurring Reelin mutation and two age-matched controls. The mutants carried a 31 bp deletion in the RELN gene that produces a premature termination codon and the loss of the Reelin protein 25 . Sheep were sampled by cerebellomedullary cistern puncture at the Veterinary Faculty of Leon, Spain, by qualified veterinarians following standard procedures 52 and conducted under license issued in accordance with European Union legislation (European Community Directive, 86/609/EC and Directive 2010/63/EU of the European Parliament and of the Council). Experimental protocols were approved by the ethic committee of Universidad de Leon, Spain. All animals were managed in accordance with the guidelines for the accommodation and care of animals.
Reelin and ApoER2 qRT-PCR analysis. RNA was extracted from human brains using the TRIzol ® Reagent in the PureLink ™ Micro-to-Midi Total RNA Purification System (Life Technologies) following the manufacturer's instructions. SuperScript ™ III Reverse Transcriptase (Life Technologies) was used to synthesize cDNAs from this total RNA (2 μ g) using random primers according to the manufacturer's instructions.
Quantitative PCR amplification was performed on a StepOne ™ Real-Time PCR System (Applied Biosystems, Life Technologies) with TaqMan probes specific for human RELN (assay ID: HS01022646_m1 Applied Biosystems), human ApoER2 (assay ID: HS00182998_m1 Applied Biosystems) and human GAPDH as an endogenous controls (Applied Biosystems). The Reelin and ApoER2 transcripts were quantified using the relative standard curve method normalized to GAPDH from the same cDNA preparation, to confirm the specificity of the PCR products from the dissociation curves.
Statistical analysis. All data were analyzed by one-way analysis of variance (ANOVA), or through a Student's t test (two-tailed) for single pair-wise comparisons, determining the exact p values. When normality Scientific RepoRts | 6:31646 | DOI: 10.1038/srep31646 was rejected, a Mann-Whitney Rank Sum Test was used. The results are presented as the means ± SEM and all the analyses were all performed using SigmaStat (Version 2.0; SPSS Inc.). Correlations between variables were assessed by linear regression analyses. A p value < 0.05 was considered significant.