Reelin expression and glycosylation patterns are
altered in Alzheimer’s disease
Arancha Botella-Lo ´pez*, Ferran Burgaya†, Rosalina Gavı ´n†, M. Salud Garcı ´a-Ayllo ´n*, Estrella Go ´mez-Tortosa‡,
Jordi Pen ˜a-Casanova§, Jesu ´s M. Uren ˜a†, Jose ´ A. Del Rı ´o†, Rafael Blesa¶, Eduardo Soriano†, and Javier Sa ´ez-Valero*?
*Instituto de Neurociencias de Alicante, Universidad Miguel Herna ´ndez-Consejo Superior de Investigaciones Cientı ´ficas, E-03550 Sant Joan d’Alacant, Spain;
†Institut de Recerca Biome `dica?Parc Cientific de Barcelona and Department of Cell Biology, University of Barcelona, E-08028 Barcelona, Spain;‡Neurology
Department, Fundacio ´n Jime ´nez Dı ´az, Brain Bank for Neurological Research, Complutense University, E-28040 Madrid, Spain;§Neurology Department,
Hospital del Mar, Institut Municipal d’Assistencia Sanitaria, E-08003 Barcelona, Spain; and¶Neurology Department, Hospital de la Santa Creu i Sant Pau,
E-08025 Barcelona, Spain
Communicated by Erminio Costa, University of Illinois, Chicago, IL, February 15, 2006 (received for review September 16, 2005)
Reelin is a glycoprotein that is essential for the correct cytoarchi-
tectonic organization of the developing CNS. Its function in the
adult brain is less understood, although it has been proposed that
Reelin is involved in signaling pathways linked to neurodegenera-
tion. Here we analyzed Reelin expression in brains and cerebro-
spinal fluid (CSF) from Alzheimer’s disease (AD) patients and
nondemented controls. We found a 40% increase in the Reelin
protein levels in the cortex of AD patients compared with controls.
Similar increases were detected at the Reelin mRNA transcriptional
level. This expression correlates with parallel increases in CSF but
not in plasma samples. Next, we examined whether CSF Reelin
levels were also altered in neurological diseases, including fron-
totemporal dementia, progressive supranuclear palsy, and Parkin-
son’s disease. The Reelin 180-kDa band increased in all of the
neurodegenerative disorders analyzed. Moreover, the 180-kDa
Reelin levels correlated positively with Tau protein in CSF. Finally,
we studied the pattern of Reelin glycosylation by using several
lectins and the anti-HNK-1 antibody. Glycosylation differed in
plasma and CSF. Furthermore, the pattern of Reelin lectin binding
differed between the CSF of controls and in AD. Our results show
that Reelin is up-regulated in the brain and CSF in several neuro-
cellular origins, thereby supporting that Reelin is involved in the
pathogenesis of a number of neurodegenerative diseases.
HNK-1 ? neurodegeneration ? cerebrospinal fluid ? blood ? biomarker
very-low-density lipoprotein receptor (1, 2), which transduce the
Reelin signal through the intracellular adapter disabled-1 (3–5).
Reelin signaling triggers a disabled-1-dependent signaling cas-
cade involving several kinases, which ultimately controls proper
neuronal migration and positioning during CNS development
(for review see ref. 6).
The complex pattern of Reelin expression is consistent with
evidence that this protein has multiple roles in brain development
and adult brain function (7–9). In the adult mammalian brain,
Reelin has been proposed to influence synaptogenesis and neural
plasticity and to favor memory formation (8–12). Reelin is also
expressed in peripheral tissues, including the liver, and is detected
in blood (10, 13). However, whether brain and other tissues
contribute to the pool of Reelin in blood remains to be elucidated.
In this context, we recently reported the presence of detectable
levels of Reelin in adult cerebrospinal fluid (CSF) (14).
Furthermore, the involvement of the Reelin signaling pathway
in neurodegeneration has also been proposed (1, 6, 9, 15–17).
First, Reelin binds to apolipoprotein E (ApoE) receptors, and
some ApoE gene polymorphisms are considered risk factors for
Alzheimer’s disease (AD). Moreover, the lack of Reelin is
associated with increased phosphorylation of Tau (1, 2, 18),
whose hyperphosphorylation leads to intracellular tangles and
neuronal degeneration (19). Disabled-1 binds to ?-amyloid
eelin is an extracellular 420-kDa glycoprotein that binds to
the transmembrane receptors apolipoprotein receptor 2 and
precursor protein (20, 21) family members, whose proteolytic
processing leads to the A?-peptide-forming amyloid plaques.
Recent data also indicate that Reelin is present in ?-amyloid
plaques in a transgenic mouse model of AD (22).
Reelin is cleaved in vivo at two sites, which results in the
production of several fragments whose relative abundance dif-
fers in distinct tissues (13, 23). We previously demonstrated the
presence of the three Reelin bands (full-length 420-kDa, 310-
kDa, and 180-kDa fragments) in human CSF and a significant
increase in the 180-kDa band in a cohort of AD patients as
compared with control individuals (14). Here we further cor-
roborate these findings and show that increased Reelin levels in
CSF of AD patients correlate with augmented brain expression
of Reelin at mRNA and protein levels. We also show that Reelin
levels in CSF are increased in other neurological disorders
including frontotemporal dementia, progressive supranuclear
palsy, and Parkinson’s disease (PD). These findings indicate that
Reelin may be a reliable molecular marker for neurodegenera-
tive diseases. Finally, we show that the pattern of Reelin
glycosylation in CSF and plasma differ, which indicates a distinct
Western Blot Analysis of CSF Reelin in AD. To confirm whether
Reelin levels are altered in AD CSF, we analyzed Reelin
expression in 19 AD patients and 11 nondemented controls
(NDC) (Fig. 1A). Because methodological handling and storage
conditions may influence the processing of Reelin (see ref. 24
and Fig. 6, which is published as supporting information on the
PNAS web site), all analyses were performed in samples frozen
at ?80°C, thawing-freezing cycles were avoided, and heating
before electrophoresis was limited to 3 min. Three typical
Reelin-immunoreactive bands were observed in all CSF analyses
by using the 142 antibody, in agreement with previous studies
(14, 25, 26). In all cases, the 180-kDa fragment was more
abundant than the 310-kDa and 420-kDa bands, the latter
corresponding to full-length Reelin. A significant ?40% in-
compared with NDC subjects (P ? 0.001; Fig. 1B). Moreover, 15
of 19 AD cases had values ?0.62 (arbitrary densitometric units)
for this band, whereas only 2 of 11 controls were above this value
Increased 180-kDa Reelin immunoreactivity may not neces-
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
Abbreviations: AD, Alzheimer’s disease; ApoE, apolipoprotein E; CSF, cerebrospinal fluid;
PD, Parkinson’s disease; Con A, Canavalia ensiformis lectin; LCA, Lens culinaris agglutinin;
?To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0601279103 PNAS ?
April 4, 2006 ?
vol. 103 ?
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sarily indicate increased full-length Reelin abundance, but in-
creased Reelin proteolytic processing of Reelin. To determine
whether the increased 180-kDa band reflected increased protein
processing, we compared the concentrations of all three Reelin
fragments in CSF. Our analyses revealed that the levels of the
420-kDa and 310-kDa bands were similar in AD and NDC cases
(Fig. 1C). These observations confirm our previous findings and
indicate that total Reelin levels are increased in the CSF of AD
patients and mostly correspond to augmented intensity of the
Expression of Reelin in Brains of AD and Control Subjects. To deter-
mine whether changes in CSF Reelin reflect differences in brain
levels of this protein, we examined the expression of Reelin in
tissue samples from the frontal cortex and cerebellum of AD and
NDC cases. In agreement with previous reports (27, 28), the
bands, similar to CSF samples. The major 180-kDa Reelin
fragment was detected in all samples, with the 420-kDa and
310-kDa bands being faintly stained in most cases (Fig. 2A).
Quantitative analyses showed a significant increase in both the
180-kDa band (P ? 0.001; a 33% increase) and total Reelin
content (the sum of the three bands; P ? 0.001; a 40% increase)
in the frontal cortex of AD patients compared with NDC
subjects (Fig. 2C). In contrast, we found similar Reelin levels in
the cerebellum of AD and NDC subjects (Fig. 2 B and D). These
results show that Reelin levels in CSF parallel protein expression
in brain areas targeted by AD.
A semiquantitative PCR assay was further designed to deter-
mine whether changes in Reelin protein corresponded to alter-
ations in mRNA expression. mRNA was purified from the
frontal cortex and cerebellum of the same cases described above,
retrotranscribed, and PCR-amplified by using33P-dATP as a
tracer and a low number of cycles to guarantee a scalar cDNA
amplification. Identical findings were observed in three inde-
pendent assays. The gels shown in Fig. 3A exhibit an apparent
increase in Reelin cDNA content in the AD frontal cortex
compared with controls. Reelin mRNA levels showed a signif-
icant (64%) increase in AD patients (levels normalized with
respect to GADPH mRNA; Fig. 3C). In contrast, Reelin mRNA
expression was not altered in samples from the cerebellum of the
indicate a transcription alteration of the Reelin gene in cortical
regions affected by AD.
Western Blot Analysis of Plasma Reelin in Subjects with AD. To
determine whether the Reelin increase in CSF of AD cases is
also detected in other biopsy fluids, we analyzed the levels of this
protein in the plasma of 9 AD patients, 12 subjects with mild
cognitive impairment, and 44 NDC subjects (Fig. 7, which is
published as supporting information on the PNAS web site).
Similar to previous reports (13, 24), the relative abundance of
Reelin bands differed in plasma and CSF. Neither the intensity
of the individual bands nor their relative banding pattern was
altered in plasma from AD and mild cognitive impairment
patients compared with NDC cases. These findings show that the
increased Reelin expression in brain tissue and CSF samples of
AD patients does not correlate with plasma levels of this protein.
Western Blot Analysis of CSF Reelin in Other Neurological Disorders.
In a previous report we found that the 180-kDa Reelin band
was also increased in the CSF of frontotemporal dementia
patients compared with controls (14). To assess whether
Reelin levels in CSF are increased in other pathological
disorders, we examined the levels of this protein in CSF
samples from several neurological diseases (Fig. 8, which is
(see Supporting Text).*, Significantly different (P ? 0.05) from the NDC group as assessed by Student’s t test. Immunoreactive bands are also shown. (C) Scatter
plots for the full-length 420-kDa and 310-kDa Reelin fragments. Because the predominant 180-kDa band displayed greater immunoreactivity than the 410-kDa
and 320-kDa fragments, semiquantitative analysis of all the fragments was performed over a range of exposure times to avoid loss of linearity during the long
exposure times required for the detection of the large fragments. Accumulative immunoreactivity from the sum of higher-molecular-mass Reelin bands is also
Immunodetection of CSF Reelin. (A) Representative blot of Reelin in CSF samples from AD and NDC. (B) Scatter plots for 180-kDa Reelin. The dashed
www.pnas.org?cgi?doi?10.1073?pnas.0601279103 Botella-Lo ´pez et al.
published as supporting information on the PNAS web site).
Significant increases in Reelin were detected in the 180-kDa
band in samples from frontotemporal dementia, progressive
supranuclear palsy, and PD patients, similar to those observed
in AD cases. Once again, the concentrations of the full-length
420-kDa Reelin and 310-kDa bands did not differ significantly
from controls (data not shown). We thus conclude that the
180-kDa Reelin band increases significantly in the CSF of all
of the neurodegenerative diseases analyzed.
Because Tau protein is a well established CSF marker of
many neurodegenerative disorders, we next compared Tau and
Reelin levels in CSF samples (Fig. 9, which is published as
supporting information on the PNAS web site). Interestingly,
there was a positive correlation between Tau levels and the
intensity of the 180-kDa band in AD patients and NDC cases
(r ? 0.66 and P ? 0.006, linear regression), which was
maintained when all samples were analyzed together (r ? 0.55
and P ? 0.001).
and cerebellum extracts (B). In each determination (made in triplicate) protein was adjusted to ?20 ?g by lane, and ?-tubulin (1:1,000; Sigma) immunoreactive
intensity was used as a control of blotting efficiency (Lower). (C and D) Reelin immunoreactivity from the 180-kDa fragment, and accumulative from the three
Reelin bands, for NDC and AD subjects in frontal cortex (C) and cerebellum (D). In one NDC sample, no higher-molecular-mass Reelin fragments were detected
and estimated in frontal cortex. The data represent the means ? SE (determinations by duplicate).*, Significantly different (P ? 0.05) from the NDC group.
Concentrations and gel mobility for Reelin fragments in AD and NDC brain. (A and B) Three Reelin bands at 420, 310, and 180 kDa in frontal cortex (A)
mixed in a single tube and electrophoretically separated in 4% acrylamide gels. The amplified bands were identifed according to their sizes (513 bp for Reelin
from frontal cortex (C) and cerebellar extracts (D).*, Significantly different (P ? 0.05) from the NDC group.
RNA expression in AD cerebral tissue. (A and B) RT-PCR-amplified cDNAs of Reelin and GAPDH from frontal cortex (A) and cerebellum (B) extracts were
Botella-Lo ´pez et al. PNAS ?
April 4, 2006 ?
vol. 103 ?
no. 14 ?
Lectin Binding and HNK-1 Immunoreactivity of CSF and Plasma Indi-
cate Distinct Reelin Origins.TheabovedataindicatedthatCSFand
plasma Reelin might originate in distinct tissues. To compare the
pattern of Reelin glycosylation in CSF and plasma, samples from
five NDC cases were incubated with several immobilized lectins
[Canavalia ensiformis lectin (Con A), Lens culinaris agglutinin
(LCA), Ricinus communis agglutinin (RCA120), and Triticum
vulgaris agglutinin (wheat germ, WGA)] (Fig. 4A) that recognize
distinct sugar sequences (29). Plasma Reelin was bound strongly
to Con A, RCA, and WGA lectins and more weakly to LCA. All
three Reelin fragments exhibited this pattern of labeling. In
contrast, 420-kDa Reelin in CSF was strongly recognized by Con
A and WGA, but also by LCA, whereas binding was weaker for
RCA. Indeed, in contrast to CSF samples, RCA binding was
conspicuous for the 180-kDa band in plasma, whereas this band
was poorly recognized by LCA and WGA. These differences in
the binding properties of plasma and CSF Reelin to lectins
indicate a distinct pattern of glycosylation and thus a different
cellular origin of Reelin protein in these two fluids.
To study whether Reelin glycosylation is altered in AD, we
further analyzed the pattern of glycosylation in CSF from NDC
and AD subjects (Fig. 4B). There was a small but significant
difference in the binding of the 180-kDa Reelin to LCA (P ?
0.02) between the AD group and controls and a nonstatistically
significant increase in the proportion of 180-kDa Reelin that
does not bind to Con A (P ? 0.06) in AD samples compared with
the NDCs. We defined a quotient LCA?Con A as the 180-kDa
Reelin unbound to LCA divided by the 180-kDa Reelin unbound
to Con A for each sample, which provided greater discrimination
between the two groups (P ? 0.008; Fig. 4C). With this analysis,
10 of 11 samples from cases with AD were below an arbitrary
cutoff point, whereas seven of the nine CSF samples from
control cases were above this cutoff.
The HNK-1 glycoepitope, an unusual 3?-sulfated glucuronic
acid sequence characteristic of neural recognition molecules,
serves as a ligand in cell interactions (30). To determine whether
Reelin contains HNK-1, we processed for Western blot G10-
immunoprecipitated Reelin using the anti-HNK-1 antibody.
Immunoprecipitation was confirmed by Western blotting by
using the anti-Reelin 142 antibody (Fig. 5). The 180-kDa band
We also identified a faint HNK-1 band in the 180-kDa Reelin
band of CSF samples but not in plasma samples. These results
further indicate that brain?CSF and plasma Reelin have essen-
tially distinct cellular origins. Moreover, the presence of the
HNK-1 epitope in brain Reelin opens up the possibility that this
sequence may participate in Reelin functions in the brain.
Reelin Levels Are Increased in the CSF and Brain, but Not Plasma, of
AD Patients. Several studies have reported decreased Reelin
mRNA and protein levels in brains in several psychiatric disor-
ders, including schizophrenia, bipolar disorder, and autism,
which were correlated with decreased plasma protein levels (27,
28, 31). Recent evidence indicates that this is mainly caused by
alterations in the methylation of the Reelin gene, which may lead
to altered Reelin gene expression throughout the body (32). Our
previous study showed detectable levels of Reelin in CSF and
increased levels of the 180-kDa fragment in AD patients (14).
Detection of Reelin in CSF has also been reported in another
study (26). Here we used a cohort of AD samples, analyzed with
the 142 anti-Reelin antibody, which exhibits a high affinity for
human Reelin (13, 33). We detected the 420-kDa, 310-kDa, and
180-kDa Reelin bands, confirming a marked increase in the
shorter fragment in the CSF and brains of AD. Although a
differential proteolytic processing of Reelin in AD may also
contribute to the increase in the 180-kDa band, the observation
that the higher-molecular-mass Reelin bands are constant in AD
and NDC indicates a net increase in Reelin protein abundance
We also found that the pattern of Reelin-lectin binding is
altered for two mannose-specific lectins, LCA and Con A.
Several neuropathological disorders, including AD, cause char-
acteristic changes in the glycosylation pattern of proteins (34),
which may be due to alterations in the contribution of different
cell types to the protein pool, imbalance of protein glycoforms,
CSF and plasma samples were incubated with immobilized lectins Con A, LCA,
WGA, and RCA. Attempts to assay the Reelin bound to each lectin by resus-
pending and boiling the resin showed only 60–80% of recovery of the
glycoprotein. The unbound Reelin, assayed by Western blotting, in the super-
natant fraction was therefore used to compare differences in lectin binding
between groups. The data represent the percentages of bound Reelin calcu-
lated after subtraction of unbound immunoreactivity for each band. We
grouped values into four categories: ???, 75–100% of Reelin binding to
and 11 AD subjects. (C) Scatter plots for the 180-kDa Reelin LCA?Con A ratio
dashed line represents an arbitrary cutoff that maximally discriminated be-
tween AD and NDC groups (?35 arbitrary units). The data represent the
means ? SE.*, Significantly different (P ? 0.05) from the NDC group as
assessed by Student’s t test.
Glycosylation of Reelin in CSF and plasma. (A) Five nonpathological
using the G10 antibody (lanes 2, 4, and 6). The experiments were repeated
three times. Western blot analyses were performed by using 142 (anti-Reelin;
A) and Ab2 (anti-HNK-1; B) antibodies. B shows the immunoreactivity of the
180-kDa cortical frontex (lane 2) for HNK-1 and more weakly for CSF-Reelin
(lane 4). In contrast, no HNK-1 band was detected in Reelin immunoprecipi-
tated from plasma (lane 6).
Reelin carries the HNK-1 carbohydrate. Reelin from frontal cortex
www.pnas.org?cgi?doi?10.1073?pnas.0601279103Botella-Lo ´pez et al.
or altered glycosylation mechanism. The cellular origin of the
abnormally glycosylated Reelin and whether the altered glyco-
sylation pattern in AD is a direct consequence of altered
metabolism or reflects changes in differentiation state warrant
In a recent report Ignatova et al. (26) failed to confirm altered
levels of the 180-kDa Reelin fragment in AD samples. We
believe that either the reduced sample size or the handling of the
samples may have contributed to the divergent results. Here we
report consistent evidence not only of increased Reelin levels in
CSF and affected brain areas of AD patients but also of
increased transcriptional activity of the Reelin gene. Moreover,
our data show that CSF storage and heating conditions are key
factors in determining Reelin protein levels, in agreement with
another study (24). Our covariance analyses further show that
these differences are not attributable to age or gender (see
Supporting Text and Table 1, which are published as supporting
information on the PNAS web site).
Reelin as a Marker of Neurodegenerative Diseases. To examine
whether Reelin could be used as a tool in the differential
we also analyzed CSF Reelin levels in samples from frontotem-
poral dementia, progressive supranuclear palsy, and PD subjects.
We found a significant increase in the levels of this protein in the
CSF of all these neurodegenerative diseases when compared
with controls. Furthermore, Reelin levels were not substantially
different among the distinct diseases, indicating that increased
expression of this protein in this fluid is not disease-specific. Our
findings indicate that increased Reelin levels in CSF could be
considered a general marker for neurodegenerative diseases.
Whether this reflects the participation of Reelin in the patho-
genesis of these diseases or is secondary to the degenerative
process itself remains to be elucidated.
CSF and Plasma Reelin Are Likely to Be Synthesized in Distinct Tissues.
Because of the lack of efficient biomarkers for neurodegenera-
tive disorders in blood, here we studied whether plasma Reelin
levels correlate with AD. We did not detect major changes in
Reelin protein levels in plasma, in contrast to the data in CSF
and brain. These results imply that Reelin detection in plasma is
not an efficient prognostic marker for CNS neurodegenerative
disorders and indicate that plasma and CSF Reelin may have
distinct cell origins.
In addition to the CNS, Reelin is expressed in other organs
including kidney, liver, and adrenal and pituitary glands (10, 13).
We found that Reelin is differentially glycosylated in CSF and
plasma, as detected with several lectins. Moreover, only brain
and CSF Reelin reacts with the Ab-2 antibody recognizing the
glycoepitope HNK-1. Because it is believed that different cell
types add distinct carbohydrate moieties to the same glycopro-
teins, our results indicate that Reelin in these two fluids mainly
reflects their expression and secretion by distinct cell types. The
observations that Reelin levels in CSF correlate with those in
brain and that Reelin is probably not expressed in human
choroid plexus cells (unpublished observations) indicate that
CSF Reelin is largely originated in brain. However, we cannot
discard a small contribution of brain Reelin to plasma Reelin
levels, because the Reelin receptor apolipoprotein receptor 2 is
expressed by choroid plexus cells (35, 36).
Reelin is likely to play a role in long-term synaptic plasticity
in adulthood by activating signaling cascades similar to those
acting in neural development (37). The expression of the rec-
ognition molecule-associated carbohydrate HNK-1 in Reelin
raises the possibility that this epitope may contribute to Reelin
function. HNK-1 expression in molecules such as integrins,
proteoglycans, and glycolipids is believed to be involved in the
functional fine-tuning of these molecules in processes such as
migration, cell aggregation, and differentiation (30, 38), pro-
cesses in which Reelin has been implicated.
Reelin and Neurodegeneration in AD. The observation of marked
increases in Reelin protein levels in brains from AD patients,
accompanied by a similar increase in Reelin transcripts,
further supports the notion that the levels of this protein are
intrinsically altered in this pathology. Furthermore, Reelin is
overexpressed in cortical regions targeted by AD but not in the
cerebella of the same patients. These observations, together
with the close correlation of the 180-kDa Reelin band levels
and Tau protein in CSF, indicate that altered expression of
Reelin and abnormal Reelin signaling may participate in the
pathogenesis of AD. The molecular mechanisms by which
Reelin contributes to AD are not known. In this context, recent
data have shown the presence of Reelin associated with
amyloid plaques in a transgenic AD mouse model (22).
Although alterations in Reelin-positive Cajal–Retzius cells in
AD appear to be conflictive (39, 40), the expression of Reelin
in cortical interneurons has not been investigated. Moreover,
Reelin binds to ApoE receptors, and several downstream
targets of the Reelin pathway are mediators of Tau hyper-
phosphorylation (2). In this context, it is interesting to note
that Reelin signaling is believed to control both cyclin-
dependent kinase 5 and glycogen synthase kinase 3, two key
kinases that regulate Tau phosphorylation in AD (41–43).
Furthermore, the in vitro interaction of Reelin with lipoprotein
receptors is inhibited in the presence of ApoE3 and ApoE4
alleles (1), the latter being genetically associated with late-
onset AD (44). The presence of ApoE also reduces Tau protein
phosphorylation protein kinase activity in Reelin-deficient
In summary, the present findings show that the expression of
the Reelin gene and Reelin protein is increased in brains of AD
patients. This expression correlates with similar increases in CSF
but not in plasma samples, indicating that Reelin has distinct cell
origins in these two tissue fluids. Finally, our data indicate that
Reelin overexpression in CSF may be a general biomarker for
several neurodegenerative diseases, although the precise mech-
anisms through which Reelin participates in the pathogenesis of
these disorders require further study.
Materials and Methods
Preparation of Samples. For diagnosis criteria see Supporting Text.
CSF and plasma aliquot samples were stored at ?80°C until their
use. Pieces (0.2 g) of human frontal cortex and cerebellum (same
cases) stored at ?80°C were thawed slowly at 4°C and homog-
enized (10% wt?vol) in 50 mM Tris-HCl, pH 7.4?150 mM
NaCl?0.5% Triton X-100?0.5% Nonidet P-40 and a mixture of
proteinase inhibitors (34). The homogenates were sonicated and
centrifuged at 20,000 ? g at 4°C for 20 min, and the supernatant
was collected and frozen at ?80°C until assays.
Western Blot Analysis of Reelin. Reelin was determined essentially
as described (24). Briefly, CSF (50 ?l), plasma (1.2 ?l), and brain
(20 ?g) samples were analyzed on 6% SDS resolving gels. The
sample boiling time was minimized to 3 min, and electrophoresis
was allowed to proceed at a voltage that prevented excessive
heat. Proteins were blotted onto nitrocellulose membranes
(Schleicher & Schuell). Blots were blocked and incubated in
monoclonal mouse anti-Reelin antibody 142 (1:200 dilution;
Chemicon International; see ref. 33). Reelin was detected by
ECL by using the ECL-Plus kit (Amersham Pharmacia). Two
control samples were used to normalize immunoreactive Reelin
signal. For semiquantitative studies, the intensity of Reelin
bands was measured by densitometry by using SCIENCE LAB
IMAGE GAUGE V4.0 software provided by Fuji Photo Film.
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April 4, 2006 ?
vol. 103 ?
no. 14 ?
Semiquantitative PCR Assay for Reelin RNA. Total RNA from brain Download full-text
tissue was purified by using an SV Total RNA purification kit
(Promega), and 2 ?g of RNA was retrotranscribed by adding the
antisense oligonucleotides to be used in the PCR assays instead
of oligo(dT). This assay consisted essentially of the same meth-
odology reported by A ´lvarez-Dolado et al. (45) for the quanti-
fication of Reelin mRNA (see Supporting Text).
Lectin Binding Analysis of Reelin. Aliquots (100 ?l) of CSF and
plasma (diluted 1:20 in PBS) were mixed with 40 ?l of immo-
bilized lectins (Sigma) and incubated overnight at 4°C. Unbound
Reelin was separated by centrifugation and examined by West-
Reelin Immunoprecipitation and HNK-1 Western Blot Analysis. Brain
(frontal cortex), CSF, and plasma (diluted 1:2 and 1:20, respec-
tively, in RIPA buffer) samples from NDC subjects were pread-
sorbed to protein A Sepharose (Amersham Pharmacia Bio-
sciences) and incubated with the anti-Reelin G10 antibody
(Chemicon International; see ref. 33). Immune complexes were
precipitated by adding protein A Sepharose. The supernatant
was collected, and the resin was washed, resuspended, and boiled
in SDS?PAGE sample buffer. Reelin was detected by using the
142 antibody, and HNK-1 was detected by using the mouse Ab-2
antibody (1:500 dilution; Neomarkers Lab Vision).
We thank Dr. A. M. Goffinet (University of Louvain Medical School,
Brussels) for the generous gift of Reelin antibodies used during prelim-
inary experiments. J.S.-V. was supported by grants from La Caixa
Foundation and the Fondo de Investigaciones Sanitarias Grant 03?0038;
E.S. and R.B. were supported by La Caixa Foundation, the Pfizer
Foundation, La Marato ´ TV3, and Ministerio de Ciencia y Tecnologı ´a
Grant SAF94-07929; J.M.U. was supported by Fondo de Investigaciones
Sanitarias Grant 04?2280; and J.A.D.R. was supported by Ministerio de
Ciencia y Tecnologı ´a Grant BFI2003-03594.
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