Received: 29 July 2022 Revised: 3 March 2023 Accepted: 19 March 2023

DOI: 10.1111/ejn.15971

RESEARCH REPORT

A novel Reelin construct, R36, recovered behavioural

deficits in the heterozygous reeler mouse

Nicole K. Morrill | Qingyou Li | Aurelie Joly-Amado | Edwin J. Weeber |

Kevin R. Nash

Department of Molecular Pharmacology

and Physiology, Morsani College of
Medicine, University of South Florida,
Tampa, FL, USA
Correspondence
Kevin R. Nash, Department of Molecular
Pharmacology and Physiology, Morsani
College of Medicine, University of South
Florida, 12901 Bruce B. Downs Blvd.,
Tampa, FL 33612, USA.
Email: nash@usf.edu
Edited by: Christina Dalla
Abstract
Reelin, a large extracellular glycoprotein, plays a critical role in prenatal brain
development and postnatally in synaptic plasticity, learning and memory. Dys-
regulation of Reelin signalling has been implicated in several neuropsychiatric
disorders including schizophrenia, autism, depression and Alzheimer’s dis-
ease. Previous studies have demonstrated that Reelin’s central fragment,
R3456, binds to ApoER2, inducing ApoER2 clustering and subsequent intra-
cellular signalling. We previously reported the development of a novel lucifer-
ase complementation assay, which we used to demonstrate that R3456 can
lead to ApoER2 receptor dimerization. Using this same assay, we explored var-
ious smaller fragments and combinations from R3456, and we identified a con-
struct of repeats 3 and 6 (R36), which could still elicit equivalent receptor
dimerization. The purpose of this study was to test R36 for biological effects
in vitro and in vivo. We show that R36 was capable of initiating intracellular
signalling in primary neuronal cultures. In addition, we demonstrate that a
single intracerebroventricular injection of R36 protein into a model of Reelin
deficiency, the heterozygous reeler mice, can significantly improve cognition.
These data support a role for the new construct R36 to enhance the Reelin
pathway, and the future possibility of exploring gene therapy approaches with
R36 in diseases characterized by reduced levels of Reelin.

KEYWORDS
HRM model, Reelin, Reelin supplementation, therapeutics

Abbreviations: AKT, protein kinase B; AMPA, α-amino-3-hydroxy-
5-methyl-4-isoxazolepropionic acid; ApoER2, apolipoproten E receptor
2; CDK, cyclin-dependent kinase; Dab1, Disabled-1; EGF, Epidermal
growth factor; ERK, extracellular signal-regulated kinase; GABA,
Gamma-aminobutyric acid; GSK3β, glycogen synthase kinase 3-beta;
HRM, heterozygous reeler mouse; ICV, intracerebroventricular; NMDA,
N-methyl-D-aspartate; VLDLR, very low density lipoprotein receptor.
Nicole K. Morrill and Qingyou Li are co-first authors.
© 2023 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.
1 | INTRODUCTION
Reelin is a large glycoprotein (450 kDa) and contains an
F-spondin homology region located at the N-terminus,
eight repeats and a small positively charged C-terminus
(Figure 1a) (D’Arcangelo et al., 1995, 1997). Each repeat
contains two subdomains A and B, separated by an epi-
dermal growth factor (EGF)-like motif (D’Arcangelo
et al., 1997; Jossin et al., 2004; Royaux et al., 1997). Reelin

Eur J Neurosci. 2023;57:1657–1670. wileyonlinelibrary.com/journal/ejn 1657

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1658 MORRILL ET AL.

F I G U R E 1 Reelin fragments R3456 and R36 increase firefly luciferase activity. (a) Reelin is composed of eight EGF-like repeats and
undergoes cleavage at two sites as notated with black arrows, between repeats 2 and 3 and repeats 6 and 7, resulting in five naturally
occurring fragments as depicted above. This project focuses on naturally occurring repeat, R3456 and a novel made repeat, R36.
(b) HEK293T cells were co-transfected with pΔEx5-NFluc and pΔEx5-CFluc with or without pSec-R3456 or pSec-R36. Firefly luciferase
activity was increased by R3456 and R36 when compared with the ApoER2-Luc construct, F3,12 = 2.886. Data shown as mean ± SEM. For
all groups, n = 4, **p < 0.01, ****p < 0.0001.

undergoes natural cleavage at two sites, between repeats

2 and 3 and repeats 6 and 7, generating five naturally
occurring fragments (Ignatova et al., 2004; Lambert de
Rouvroit et al., 1999). Reelin can bind and signal through
two known receptors, apolipoprotein E receptor
2 (ApoER2) and very low density lipoprotein receptor
(VLDLR) (Beffert et al., 2004, 2006; D’Arcangelo
et al., 1999; Hiesberger et al., 1999; Qiu et al., 2006). Both
ApoER2 and VLDLR bind to the ligands apolipoprotein
E (ApoE) and Reelin, which can compete for receptor
binding (D’Arcangelo et al., 1999; Hiesberger
et al., 1999). It has been previously demonstrated with
immunoprecipitation experiments that the central frag-
ment containing repeats 3 through 6 or R3456 (190 kDa)
is the fragment involved in initiating the Reelin signal-
ling cascade and interacts with VLDLR and ApoER2
(Jossin et al., 2004; Royaux et al., 1997). Receptor cluster-
ing leads to the recruitment and tyrosine phosphorylation
of Disabled-1 (Dab1), via Src family kinases (Howell
et al., 1999, 2000; Strasser et al., 2004). Phosphorylation
of Dab1 leads to activation of multiple downstream sig-
nalling pathways. These pathways include activation of
the Src-family of tyrosine kinases (Arnaud et al., 2003;
Ballif et al., 2003; Jossin et al., 2003), AKT (Ballif
et al., 2003), PI3K (Bock et al., 2003; Jossin &
Goffinet, 2007), ERK (Lee et al., 2014) pathways and the
CDK pathway (Ohkubo et al., 2003; Ohshima et al., 2001;
Yamamoto et al., 2009; Yoneshima et al., 1997). This acti-
vation can lead to effects on cell adhesion, dendrite out-
growth and spine formation, as well as synaptic receptor
reorganization.
In the developing brain, Reelin plays a critical role in
guiding radial migration of neurons, whereas in adult-
hood Reelin supports synaptic plasticity and learning and
memory. Reelin is involved in potentiating glutamatergic
and GABAergic neurotransmission, aids in synaptic mat-
uration and increases AMPA and NMDA receptor expres-
sion and activity (reviewed (Jossin, 2020; Lee &
D’Arcangelo, 2016). Reductions in Reelin levels and sig-
nalling have been associated with a number of neurologi-
cal diseases including schizophrenia, autism, temporal
lobe epilepsy and Alzheimer’s disease (Cuchillo-Ibanez
et al., 2016; Fatemi et al., 2005; Folsom & Fatemi, 2013;
Ishii et al., 2016; Jossin, 2020). In humans, loss of Reelin
results in a type of lissencephaly with severe cortical and
cerebellar malformation (Hong et al., 2000). Thus, there
is a growing body of interest in finding potential thera-
peutic strategies that target Reelin signalling and supple-
mentation. The mouse reln gene spans 450 kb contains
65 exons and was first characterized in the homozygous
reeler mouse model. Anatomically, homozygous reeler
mice are characterized by abnormalities in neuronal layer
formation and positioning in brain regions during devel-
opment, leading to malformations of neocortex, cerebel-
lum and hippocampus (Boyle et al., 2011;
Hamburgh, 1963; Tissir & Goffinet, 2003). These mice
exhibit dystonic posture, tremors and a dramatic ataxic
gait, known as “reeling” phenotype as early as 2 weeks of
age, together with enhanced susceptibility to seizure
(Curran & D’Arcangelo, 1998; Falconer, 1951; Patrylo
et al., 2006; Tissir & Goffinet, 2003). Moreover, viability,
fertility and immunity are greatly reduced in these mice
(Green-Johnson et al., 1995; Meseke et al., 2018; Togo
et al., 1986). These severe deficits render difficult the use
of this model for testing of potential therapeutic targets.
The heterozygous reeler mouse (HRM) model is produced
through the heterozygous Reelin mutation resulting in a

MORRILL ET AL.
50% reduction of the Reelin protein in the CNS through-
out development and the life of the animal. Although less
severe than the homozygous reeler mice, the HRM show
biochemical alterations in spine number and morphol-
ogy, and a distinct defect in hippocampal synaptic plastic-
ity and memory dysfunction (Barr et al., 2008; Qiu
et al., 2006). Furthermore, treatment of this model with
Reelin recovered the biochemical, morphological and
physiological deficits in the adult HRM mouse. Even
more compelling was the recovery of HRM-related beha-
vioural and cognitive impairments (Rogers et al., 2013).
We have previously presented the development of a
novel luciferase complementation assay, which we used
to demonstrate that R3456 can lead to receptor dimeriza-
tion or clustering (Li et al., 2023). Here, we used this fire-
fly luciferase assay to identify smaller fragments of Reelin
which are capable of inducing receptor clustering. We
present a new fragment of Reelin that can enhance
Reelin pathway in vitro. We then test Reelin enhance-
ment through our novel Reelin fragment in the heterozy-
gous reeler mice. This study aids in supporting the
possibility of Reelin supplementation as a therapeutic
and raises the need for further work in other models
affected by a loss of Reelin expression.
2 | MATERIALS AND METHODS
2.1 | Materials
The materials used are as follows: Zeocin (InvivoGen,
ant-zn); anti-Dab1 (Millipore, AB5840); anti-pDab1
(Millipore, AB9644); anti-β Actin (Cell Signaling 4967S);
RIPA Lysis buffer (Millipore, Tris/HCl pH 7.4, 1 mM
EDTA, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycho-
late). The following were obtained from Cell Signaling:
Anti-pERK, anti-ERK; anti-Akt; anti-pAkt.
2.2 | Animal handling
Wide-type C57BL/6J (WT) mice were obtained from
Jackson laboratories and were bred for this study in
accordance with IACUC breeding protocols (Bar Harbor,
Maine, USA). Reeler heterozygous mice (HRM) (B6C3Fe
a/a-Relnrl/J; Jackson Laboratories) were generated by
heterozygous crosses and were bred for this study in
accordance with IACUC breeding protocols. Animals
were housed in standard 12-h light–dark cycle with
access to food and water ad libitum. All animal care pro-
tocols used were approved by the Institutional Animal
Care and Use Committee of the University of South Flor-
ida (PHS Assurance number: D-16-00589 [A4100-01]).
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1659
Male and female mice aged 12–14 weeks were used
within this study and the groups were as follows: HRM
given the PBS injection (5M, 5F), HRM given the R36
protein injection (5M, 5F), WT mice given the PBS injec-
tion (6M, 4F) and WT mice given the R36 protein injec-
tion (6M, 4F).
2.3 | Constructs and vector
Central fragment of human Reelin encoding amino acids
1313 to 2618 of Reelin was purified as previously
described (Li et al., 2023). The R36 construct was synthe-
sized by Gen9 Inc. (Cambridge, MA) and encompassed
the human Reelin amino acid fragments 1313–1614
(R3) and 2329–2618 (R6). R36 was ligated into the pSec-
Tag2B vector in frame with the Ig-kappa leader sequence
and the His- and Myc-tags, similar to R3456 as described
previously (Li et al., 2023).
2.4 | Production and purification of R36
A stably transfected HEK293T cell line expressing R36
was achieved with the selection reagent Zeocin
(InvivoGen) and Dot blots to screen for positive clones.
The cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% foetal bovine serum
(FBS) and 100-μg/mL Zeocin (InvivoGen) at 37 C and 5%
CO2. Reelin was purified from the conditioned medium
using a Ni-Sepharose™ Excel resin (GE) column as
described in detail previously (Li et al., 2023).
2.5 | Cell culture
For luciferase assay and transient transfections,
HEK293T cells were grown and maintained in DMEM
(Sigma-Aldrich) with 10% FBS and 1% penicillin/
streptomycin at 37 C and 5% CO2. For primary neurons,
cortical tissue including the hippocampus was isolated on
embryonic day 16 (E16) from C57BL/6 mouse pups. The
minced tissues were gently dissociated using 0.25% tryp-
sin and mechanical trituration to achieve individual cor-
tical neuronal cells. The neuronal cells were plated on a
six-well plate coated with 0.1% poly-L-lysine (Sigma-
Aldrich) at a density of 200,000 cells/well and incubated
in 3-mL of neurobasal medium (Gibco) supplemented
with 2% B-27 (Invitrogen) and 0.5-mM GlutaMax (Gibco)
at 37 C and 5% CO2. On day in vitro (DIV) 14, the
medium was replaced with neurobasal medium contain-
ing various concentrations of R36 protein (0.2 to 1 nM)
for 1 h at 37 C and 5% CO2.

1660
2.6 | Luciferase assay
Dual-Glo luciferase assay was performed in HEK293T
cells as previously described in detail in Li et al. (2023).
Cells were co-transfected with pΔEx5-NFluc and
pΔEx5-CFluc with or without pSec-R3456 or pSec-R36 to
express firefly luciferase. HEK293T cells were plated onto
a 24-well-plate at a density of 2  105 cells/well in
DMEM media supplemented with 10% foetal bovine
serum and 1% penicillin/streptomycin, and the cultures
were incubated at 37 C and 5% CO2 overnight. The fol-
lowing day, the medium was replaced by Opti-MEM
serum-free medium, and the constructs were co-
transfected with Lipofectamine 2000 (ThermoFisher).
Approximately 48 h post-transfection, the medium was
removed, and Glo Lysis Buffer (Promega) was added onto
the plate to lyse the cells for 5 min. The cells were then
centrifuged, and the supernatant and Dual-Glo Lucifer-
ase assay reagent combined in a 96-well assay plate. The
firefly luminescence was first measured by using a Bio-
Tek Synergy H4 plate reader.
2.7 | Western-blot analysis
HEK293T cells were lysed in Glo Lysis Buffer and cortical
neurons were lysed in RIPA Lysis buffer (Millipore, Tris/
HCl pH 7.4, 1-mM EDTA, 150-mM NaCl, 1% NP40,
0.25% sodium deoxycholate) containing complete prote-
ase inhibitors (Sigma) and phosphatase inhibitors I and
II (Sigma) and then centrifuged at 13,000 g for 12 min at
4 C. The supernatants were collected, and the protein
concentrations were measured using a standard BCA
assay (Pierce, Rockford, IL). Samples were prepared in
Laemmli’s sample buffer (Bio-Rad) with 5%
2-mercaptoethanol and heated for 95 C for 10-min. Pro-
teins were run on 4–15% TGX Criterion gels (Bio-Rad).
Gels were transferred to Trans-Blot® Turbo™ Midi Nitro-
cellulose blots, followed by blocking with 5% nonfat dry
milk in TBS for 1 h at room temperature. Primary anti-
bodies were incubated overnight at 4 C using the follow-
ing antibodies: Dab1 (Millipore, 1:1000), phospho-Dab1
(Abcam, 1:1000; for detecting Dab1 phosphorylated at
residue Y232), ERK1/2 (Cell Signaling, 1:2000), phospho-
ERK1/2 (Cell Signaling, 1:2000; for detecting ERK1 phos-
phorylated at residue Thr202 of ERK1 and ERK2 phos-
phorylated at Thr185/Tyr187), Akt (Cell Signaling,
1:1000), and phospho-Akt (Cell Signaling, 1:1000; for
detecting phosphorylation of Akt at Ser473), GSK-3β
(Cell Signaling [3D10 Cat#9832], 1:1000), phospho-GSK-
3β (Cell Signaling [D85E12, Cat# 5558], 1:1000). Actin
(Sigma, 1:3000) was used as a protein control. Mem-
branes were washed 3  10 min in TBS with 0.1% Tween
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MORRILL ET AL.
20 prior to incubation with the appropriate secondary
antibody with HRP-conjugation (GE, anti-mouse IgG and
anti-rabbit IgG 1:3000) for 1 h at room temperature. Fol-
lowing a repeated washing, the blots were developed with
an enhanced chemiluminescence reagent
(ThermoFisher), then exposed to an X-ray film which
was quantified by using Image J software (NIH).
Western Blot analysis of pre- and post-synaptic
markers: Dissected mouse brain regions were homoge-
nized in RIPA buffer for western blot analysis as detailed
above. We used the following primary antibodies: anti-
post-synaptic density protein-95 (PSD95) antibody
(Abcam; 1:500) and anti-synaptophysin antibody
(Abcam; 1:1000). Revert total protein (LI-COR) was used
as a protein control. Membranes were washed for 10 min,
3X in TBS with 0.1% Tween 20 followed by incubation
with fluorescently tagged secondary antibody (anti-
mouse IgG and anti-rabbit IgG, 1:10,000, respectively) for
1 h. Proceeding washing, the blots were scanned and
quantified using an Odyssey Dlx (LI-COR biosciences)
scanner and software.
2.8 | Direct bilateral ventricular
injections
Mice were anaesthetized with isoflurane and placed on a
stereotaxic surgery apparatus (World Precision Instru-
ments). A subcutaneous injection of Nocita (bupivacaine
liposome injectable suspension, Elanco) 20 μL at
13.3 mg/mL was performed on the head of the animal as
a long-lasting post-surgical pain analgesic. After 2 min, a
sagittal incision was made mid-cranium and the skin
gently pushed back to enlarge the opening. Two holes
were drilled through the skull to allow passage of the
Hamilton needle through the brain, into the ventricles
(AP 0.4 mm, L ±1.0 mm, and V 2.4 mm from
bregma). Mice were injected bilaterally with 3.0-μL PBS
or 3.0 μL of Reelin (R36) at 300 nM with an injection rate
of 2.5 μL/min. This concentration of Reelin was previ-
ously used in vivo for full length Reelin protein (Rogers
et al., 2013; Weeber et al., 2002). After the injection, the
needle was left in for 2 min and then removed, and the
incision was sutured. Mice were observed post-
operatively for 2 h in individual cages on a heating pad.
Following injections, animals were allowed to recover for
2 days before beginning behavioural testing.
2.9 | Behavioural experimentation
For behaviour testing, mice were balanced for gender
and the experimentor was blinded to the treatment/

MORRILL ET AL.
genotype of the mice. Testing was performed in the fol-
lowing order: Open field, Hidden Platform water maze
and fear conditioning.
2.9.1 | Open-field
Mice were recorded in an open-field chamber
(40  40  27 cm) for 15 min. Distance travelled, move-
ment and immobility were analysed with the ANY-Maze
animal activity system (Stoelting Co.). Comparing time
spent in the perimeter versus time spent in the centre
was calculated to assess anxiety-like behaviour.
2.9.2 | Hidden platform water maze
A platform (9-cm diameter) was submerged just below
the surface in a 1.2-m diameter pool filled with water
(deep enough that mice could not touch the bottom) that
contained non-toxic white tempera paint to obscure the
platform and allow easy visibility of the mice. Bold basic
shaped visual cues were positioned around the room.
Mice were placed in the pool and allowed to swim for a
maximum of 60 s. During training, if the mice failed to
find the platform within 60 s, they were shown to
it. Training consisted of four training days with four trials
per day, separated by a 60-min inter-trial interval. On day
5, the probe trial was performed. This involved removal
of the platform and recording of the track of each
mouse’s swim pattern for 60 s with the EthoVision XT
video-tracking software (Noldus). Time spent in the
desired platform target quadrant was assessed. For plat-
form crossings, we defined a ‘crossing’ as the animal’s
centre-point entering the predefined target
platform zone.
2.9.3 | Fear conditioning
Was conducted to evaluate fear-based learning and mem-
ory. The test was conducted in a square sound-
attenuation chamber (25 cm  25 cm) with wire-grid
flooring. In training, mice were placed in the chamber
with background white noise and allowed to explore for
3 min before the conditioned stimulus was presented
(90-dB tone) for 30 s. At 28 s, the unconditioned stimulus
(a mild [0.5-mA] foot shock) was administered for a total
of 2 s. After a period of 90 s, a second conditioned stimu-
lus/unconditioned stimulus pairing was conducted, fol-
lowed by another 90 s period, for a total of 7 min.
Contextual fear conditioning was conducted 24 h after
the training in the same chamber, with white noise and
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1661
no tone, for 3 min, and freezing was assessed. Cued fear
conditioning was conducted following the contextual
trial, wherein the chamber was altered by scent, lighting
and floor texture. The mice were placed into the altered
chamber and given a 3-min habituation phase (no tone)
followed by the conditioned stimulus (90-dB tone) for the
last 3 min of the test. Freezing behaviour was recorded
via ANY-Maze software (Stoelting Co.). Freezing was
assessed as immobility for at least one consecutive
second.
2.10 | Statistical analysis
For luciferase activity measurement (Figure 1) and
western analysis of dose response to R36 (Figure 2),
one-way ANOVA analysis was performed followed by
Tukey’s Multiple Comparison post hoc test (set at a sig-
nificance of p ≤ 0.05). A two-way ANOVA followed by
Tukey’s multiple comparison post hoc test (set at a sig-
nificance of p ≤ 0.05) was used to analyse the beha-
vioural tasks and western blot data where significance
of genotype and treatment was assessed. The Grubbs
outlier test was preformed alongside each statistical
analysis using an alpha = 0.05. All statistical testing
was performed using GraphPad Prism version 9 for
MacOS (GraphPad Software, San Diego California
UDA, www.Graphpad.com).
3 | RESULTS
3.1 | Small Reelin construct, R36,
induces ApoER2 clustering
We previously described a new luciferase assay to detect
Reelin signalling (Li et al., 2023). Briefly, cells were
cotransfected with one recombinant ApoER2 receptor
fused to the N-terminus of luciferase and one ApoER2
receptor fused to the C-terminus of luciferase. We previ-
ously showed that full-length Reelin as well as central
fragment R3456 (Figure 1a) significantly increased
ApoER2 dimerization as indicated by increased firefly
luminescence activity (Li et al., 2023). In the present
study, we examined numerous small fragments of Reelin
(not shown) and found a fragment of interest, R36, con-
sisting of EGF-like domains 3 and 6 (Figure 1a). One-way
ANOVA revealed a significant increase in firefly lumines-
cence activity in presence of R36 to the same extent as
R3456 (F3,12 = 157, p = 0.00001) indicating dimerization
of ApoER2 (Figure 1b). A basal dimerization of ApoER2
was also found, consistent with our previous results (Li
et al., 2023). Tukey’s HSD test for multiple comparisons
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1662 MORRILL ET AL.

F I G U R E 2 R36 protein induces downstream signalling. Primary cortical neurons (n = 5) at DIV 14 were treated with Reelin fragment
R36 at concentration 250 pM, 500 pM, 1000 pM or none (control) for 1 h, and the cell lysates were immunoblotted. (a) Micrograph of
immunoblot probed with antibody against phosphorylated Dab1 (Y232) and against total Dab1. (b) Quantification of band intensity for
phosphorylation of Dab1 and (c) total Dab1. Results are presented as ratio to actin protein. (d) Analysis of the ratio of pDab1/Dab1.
(e) Micrograph of immunoblot probed with antibody against phosphorylated ERK1/2 and against total ERK1/2. (f) Quantification of band
intensity for phosphorylation of ERK1/2 and (g) total ERK1/2. Results are presented as ratio to actin protein. (h) Analysis of the ratio of
pERK/ERK. (i) Micrograph of immunoblot probed with antibody against phosphorylated AKT and against total AKT. (j) Quantification of
band intensity for phosphorylation of AKT and (k) total AKT. Results are presented as ratio to actin protein. (l) Analysis of the ratio of
pAKT/AKT. (m) Micrograph of immunoblot probed with antibody against phosphorylated GSK-3β and against total GSK-3β.
(n) Quantification of band intensity for phosphorylation of GSK-3β and (o) total GSK-3β. Results are presented as ratio to actin protein.
(p) Analysis of the ratio of pGSK-3β/GSK-3β. Data are shown as mean ± SEM. **p < 0.01, ****p < 0.0001.

found that the mean value of firefly luminescence activity
in presence of R36 was not different from R3456
(p = 0.70, 95% C.I. = 21,062, 45,875). Luminescence
activity observed with R36 and R3456 was significantly
increased when compared with ApoER2-luc only or con-
trol (R36 vs. control, p = 0.0001, C.I. = 235,073,
168,137; R36 vs. ApoER2luc only p = 0.0001, C.I.
= 181,653, 114,717; R3456 vs. control, p = 0.0001,
C.I. = 222,667, 155,731; R3456 vs. ApoER2luc only
p = 0.0001, C.I. = 169,247, 102,310). These results
indicate that R36, like R3456, is able to induce dimeriza-
tion of Reelin receptor ApoER2.
3.2 | R36 fragment of Reelin evokes
ApoER2-associated intracellular signalling
We then investigated if R36 could induce downstream
signalling in vitro. Primary cortical neurons were treated
with R36 at concentration 250 pM, 500 pM, 1000 pM or
none (control) for 1-h and the cell lysates were immuno-
blotted for total and phosphorylated forms of Dab1, AKT,
ERK and GSK-3β (Figure 2a,e,i,m). These proteins are
known to be involved in Reelin downstream signalling.
One-way ANOVA revealed an effect of treatment for
phosphorylated Dab1 (Figure 2b, F3,14 = 9.013,

MORRILL ET AL.
p = 0.0014) and ratio of pDab1/Dab1 (Figure 2d,
F3,15 = 4.685, p = 0.02). We found no significant effect
on total Dab1 (Figure 2c, F3,16 = 1.003, p = 0.41).
Tukey’s HSD test for multiple comparisons showed a
significant increase in phosphorylated Dab1 at high
levels of R36 (1000 pM dose) when compared with con-
trol ( p = 0.005, 95% C.I. = 0.50, 0.08), 250-pM dose
( p = 0.003, 95% C.I. = 0.61, 0.12) or 500 pM dose
( p = 0.007, 95% C.I. = 0.49, 0.07) with no significant
impact on total Dab1 or ratio of pDab1/Dab1 when the
different concentration of R36 were compared with con-
trol (Figure 2a–d).
Similarly, one-way ANOVA revealed an effect of
treatment for phosphorylated ERK1/2 (Figure 2f,
F3,16 = 24.64, p = 0.0001) and ratio of pERK/ERK
(Figure 2h, F3,16 = 18.33, p = 0.0001). We found no sig-
nificant effect on total ERK (Figure 2g, F3,16 = 0.65,
p = 0.59). Tukey’s HSD test for multiple comparisons
showed a significant increase in phosphorylated
ERK1/2 at high levels of R36 (1000 pM dose) when com-
pared with control ( p = 0.0001, 95% C.I. = 0.51,
0.26) but not for the 250-pM dose ( p = 0.24) or
500-pM dose ( p = 0.45). We found no significant
changes in the levels of total ERK1/2. The ratio of
pERK/ERK was significantly increased at high levels of
R36 (1000-pM dose) when compared with control
( p = 0.0001, 95% C.I. = 0.61, 0.27) but not for the
250-pM dose ( p = 0.42) or 500-pM dose ( p = 0.54,
Figure 2e–h).
One-way ANOVA revealed an effect of treatment for
phosphorylated AKT (Figure 2j, F3,16 = 3.88, p = 0.03),
total AKT (Figure 2k, F3,16 = 4.5, p = 0.02) and ratio of
pAKT/AKT (Figure 2l, F3,16 = 9.37, p = 0.0008). Tukey’s
HSD test for multiple comparisons failed to show a signifi-
cant difference when comparing the different doses to con-
trol for phosphorylated AKT. We found a significant
decrease in total AKT (p = 0.008, 95% C.I. = 0.06, 0.39)
and increase in the ratio of pAKT/AKT (p = 0.007, 95%
C.I. = 0.88, 0.13) when comparing high dose of R36
against control.
Finally, one-way ANOVA revealed an effect of treat-
ment for phosphorylated GSK-3β (Figure 2n, F3,16 = 5.84,
p = 0.009), and ratio of pGSK-3β/GSK-3β (Figure 2o,
F3,16 = 8.7, p = 0.001) with no effect on total GSK-3β
(Figure 2p, F3,16 = 0.94, p = 0.44). Tukey’s HSD test for
multiple comparisons failed to show a significant differ-
ence when comparing the different doses to control for
phosphorylated or total GSK-3β. We found a significant
increase in the ratio of pGSK-3β/GSK-3β (p = 0.02, 95%
C.I. = 0.68, 0.064) when comparing high dose of R36
against control.
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1663
3.3 | R36 protein injection behavioural
analysis
Next, we sought to determine if R36 had a biological
effect in vivo. We used heterozygous reeler mice (HRM)
that exhibit a 50% reduction in Reelin levels together
with cognitive deficits. As depicted in Figure 3a, HRM
and wildtype control mice aged 12–14 weeks old (n = 10/
group) were injected intracerebroventricularly with
either R36 (2-μL at 300-nM) or PBS (control). Forty eight
hours following surgery, mice underwent behavioural
testing. First, mice were tested in the open field for gen-
eral locomotor activity and anxiety like behaviour. Two-
way ANOVA showed a significant effect of genotype
(F1,36 = 7.7, p = 0.009) and treatment (F1,36 = 6.6,
p = 0.01) with no interaction (F1,36 = 3.5, p = 0.07) for
total distance travelled (Figure 3b). Tukey’s HSD test for
multiple comparisons showed a baseline locomotion
abnormality in HRM PBS injected group with reduced
total distance travelled compared with WT mice (HRM
PBS vs. WT PBS p = 0.01, 95% C.I. = 8.11, 0.80; HRM
PBS vs. WT R36 p = 0.02, 95% C.I. = 7.9, 0.60,
Figure 3b). There was no significant difference in total
time travelled between HRM mice that received the R36
protein injection and the WT animals (HRM R36 vs. WT
PBS p = 0.99, 95% C.I. = 3.85, 3.46; HRM R36 vs. WT
R36 p = 0.91, 95% C.I. = 4.52, 2.79) indicating a rescue
of this phenotype.
No differences were observed between the WT mice
injected with R36 protein and the WT control group
(p = 0.96, 95% C.I. = 4.32, 2.98, Figure 3b). Two-way
ANOVA revealed no significant difference on time in the
centre between groups (Figure 3c; genotype F1,36 = 0.25,
p = 0.62, treatment F1,36 = 0.49, p = 0.49).
Next, we examined spatial learning and memory with
the hidden platform water maze task. For all groups, the
average slope of the training graph (over the four training
days) was similar between all groups, suggesting that at
the end of training all groups had established a search
strategy to locate the platform (data not shown). During
the probe trial 24 h after training (that test for spatial
memory), two-way ANOVA revealed a significant effect
of genotype (F1,36 = 19.43, p = 0.0001) and treatment
(F1,36 = 10.34, p = 0.003) as well as interaction
(F1,36 = 6.2, p = 0.02) for time spent in quadrant
(Figure 3d). Tukey’s HSD test for multiple comparisons
revealed that HRM-PBS group spent significantly less
time in the target quadrant compared with the WT-PBS
group, demonstrating a baseline deficit in spatial learning
and memory ( p = 0.0001, 95% C.I. = 26.35, 7.61). In
contrast, the HRM-R36 group spent significantly more
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1664 MORRILL ET AL.

F I G U R E 3 R36 protein rescues locomotor and learning deficits in HRM. (a) Experimental design. (b) Total distance travelled and
(c) time in the centre, during open field test. (d) Time spent in quadrant (percentage of total time), (e) number of target platform crossing
and (f, g) micrograph representation of averaged heat maps, during Morris water maze. Percent time freezing during (h) contextual and
(i) cued fear conditioning in mice aged 12–14 weeks: HRM given the PBS injection (5M, 5F), HRM given the R36 protein injection (5M, 5F),
WT mice given the PBS injection (6M, 4F) and WT mice given the R36 protein injection (6M, 4F). Data presented as mean ± SEM. For all
groups, n = 10, *p < 0.05, **p < 0.01, ****p < 0.0001.

MORRILL ET AL.
time in the target quadrant compared with the HRM-PBS
group ( p = 0.0001, 95% C.I. = 26.35, 7.61)
(Figure 3d). The HRM-R36 group behaved similar to both
WT groups (there was no effect of treatment in the WT
group [PBS vs. R36, p = 0.96, 95% C.I. = 11.13, 7.60]).
Similarly, two-way ANOVA revealed a significant effect
of genotype (F1,36 = 4.17, p = 0.04) and treatment
(F1,36 = 5.73, p = 0.02) as well as interaction
(F1,36 = 5.73, p = 0.02) for target platform crossings
(Figure 3e). Tukey’s HSD analysis showed that PBS
injected HRM demonstrated reduced target platform
crossings compared with the WT animals (vs. WT PBS
p = 0.02, 95% C.I. = 11.71, 0.89; vs. WT R36 p = 0.02,
95% C.I. = 11.71, 0.89). Treatment with R36 rescued
this phenotype in HRM (vs. WT PBS p = 0.99, 95% C.I.
= 4.91, 5.91; vs. WT R36 p = 0.99, 95% C.I. = 4.91,
5.91; vs. HRM PBS p = 0.009, 95% C.I. = 12.21, 1.39).
Averaged heat maps are shown for each group which
corroborate visually what is observed with total time
spent in the target quadrant and total platform crossings
(Figure 3f,g). The difference in spatial memory between
the HRM group treated with PBS and the other groups is
easily visualized, with the HRM-PBS group showing no
clear quadrant preference and a search pattern encom-
passing the majority of the pool with a preference for the
edges of the pool (Figure 3g). In contrast, the HRM group
given the R36 injection demonstrates a heat map similar
to those seen for the WT groups (Figure 3f,g). These data
suggest rescue of cognitive deficits in the HRM with the
R36 protein.
Hippocampal-dependent associative learning was
assessed using a standard contextual fear conditioning
paradigm. In the contextual test, we found an effect of
genotype (F1,36 = 10.89, p = 0.002), treatment
(F1,36 = 5.92, p = 0.02) with interaction (F1,36 = 63.91,
p = 0.0001) using two-way ANOVA analysis (Figure 3h).
HRM-PBS mice showed a baseline reduction in the per-
centage of total time spent freezing when compared with
both WT groups (Tukey’s HSD test for multiple compari-
son; HRM PBS vs. WT PBS p = 0.0001, 95% C.I.
= 30.92, 14.38; HRM PBS vs. WT R36 p = 0.001, 95%
C.I. = 20.73, 4.18) (Figure 3h). The HRM-R36 group
demonstrated a significant recovery of the time spent
freezing in the contextual test when compared with the
HRM group given the PBS injection (p = 0.0001, 95%
C.I. = 30.92, 14.38, Figure 3h). Following R36 protein
injection, the HRM group performed similarly to that of
the WT groups given the PBS injection (p = 0.92, 95%
C.I. = 10.16, 10.39) suggesting that the R36 protein
injection rescued the baseline reduction in time spent
freezing during the contextual test (Figure 3h). We found
a difference between the WT-R36 group and the WT-PBS
group (p = 0.001, 95% C.I. = 19.53, 3.87) in the
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1665
contextual test, suggesting that the R36 injection
decreased the amount of freezing during contextual fear
conditioning in WT animals. Upon examining the cued-
based test, no baseline deficit was observed in the HRM
given the PBS injection and overall, no significant differ-
ences were observed amongst groups (two-way ANOVA
showed no significance for genotype: F1,36 = 1.13,
p = 0.29; or treatment F1,36 = 2.5, p = 0.12, Figure 3i).
3.4 | Western blot analysis
Following the completion of our behavioural assays, tis-
sue was collected, hippocampal brain tissue was homoge-
nized and probed for PSD95 and synaptophysin levels.
No differences were observed between all groups for
synaptophysin levels (two-way ANOVA no genotype
F1,12 = 0.38, p = 0.55 or treatment F1,12 = 0.69, p = 0.42
effect, Figure 4a,b). However, we found a genotype effect
(two-way ANOVA F1,12 = 18.34, p = 0.001) and treat-
ment effect (F1,12 = 8.6, p = 0.01) for PSD95 levels
(Figure 4c,d). There was an observed baseline deficit in
PSD95 levels for the HRM-PBS group when compared
with WT groups (vs. WT PBS p = 0.0005, 95% C.I.
= 0.89, 0.28; vs. WT R36 p = 0.001, 95% C.I. = 0.84,
0.22, Figure 4c,d). HRM that received the R36 protein
had similar levels of PSD95 when compared with WT
groups that received either the R36 protein ( p = 0.97,
95% C.I. = 0.35, 0.26) or PBS injection (p = 0.97, 95%
C.I. = 0.35, 0.26) and significantly different from HRM
PBS group ( p = 0.002, 95% C.I. = 0.79, 0.18), suggest-
ing a rescue in PSD95 levels.
4 | DISCUSSION
It has been well established that Reelin binds to two lipo-
protein receptors, VLDLR and ApoER2, causing the
receptors to cluster into dimers or higher-order multi-
mers (D’Arcangelo et al., 1999; Divekar et al., 2014;
Dlugosz et al., 2019; Hiesberger et al., 1999; Strasser
et al., 2004). Activation of Dab1 induced by ApoER2 clus-
tering upon Reelin binding is critical for Reelin intracel-
lular signalling (Bock & Herz, 2003; Bock & May, 2016;
Strasser et al., 2004) together with phosphorylation of
ERK1/2 and AKT (Lee et al., 2014; Simo et al., 2007).
Our laboratory has previously published on a novel
split-luciferase assay of ApoER2 clusterin (Li et al., 2023).
Using this assay method, we previously investigated the
Reelin fragment R3456, known to be critical for Reelin
intracellular signalling. We demonstrated that incubation
with R3456 elicited a similar response to full length
Reelin in the luciferase assay, indicating receptor

1666
dimerization (Li et al., 2023). We further identified down-
stream activation of the Reelin signalling pathway. Con-
sistent with work from others, we reported that this
central fragment of Reelin, R3456, can induce Dab1 phos-
phorylation (pDab1) in primary neurons (Jossin
et al., 2004; Lee et al., 2014; Li et al., 2023; Nogi
et al., 2006). Our recent work reveals that R3456 can also
activate ERK and AKT (Li et al., 2023). More impor-
tantly, we showed rescue of cognitive deficits in HRM
with the injection of R3456 protein.
Given our success in supplementing R3456 into the
HRM (Li et al., 2023), we wanted to further explore the
possibility of Reelin as a therapeutic to aid in disease
states affected by reduced Reelin expression and signal-
ling such as schizophrenia, autism, depression, epilepsy
and Alzheimer’s disease (Alcantara et al., 1998;
D’Arcangelo et al., 1995, 1997; Jossin, 2020; Jossin &
Goffinet, 2007; Nakajima et al., 1997). Full-length Reelin
(450 kDa) and R3456 (190 kDa) are large protein mol-
ecules which pose issues for production and cost when
considering their use as therapeutics. We hypothesized
that if there was a smaller fragment of Reelin, capable of
eliciting a similar effect as R3456 and full-length Reelin,
it could potentially offer a viable gene therapy approach.
Using our luciferase assay, we screened numerous Reelin
fragments and found that the construct consisting of
EGF-like domains 3 and 6 (R36) (Figure 1a) induced
ApoER2 dimerization or clustering (Figure 1b). Impor-
tantly, R36 also demonstrated activation of the Reelin
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MORRILL ET AL.
F I G U R E 4 R36 protein injection
enhances markers of post-synaptic
density in the HRM. (a) Anti-
synaptophysin western blot of
hippocampal samples from WT and
HRM groups that received PBS or R36
protein injections. (b) Quantification of
western blot probed for anti-
synaptophysin. Results are presented as
ratio to revert total protein. (c) Anti-
PSD95 western blot of hippocampal
samples from WT or HRM groups that
received PBS or R36 protein injections.
(d) Quantification of western blot
probed for anti-PSD95. Results are
presented as ratio to revert total protein.
n = 4, **p < 0.01. Data shown as mean
± SEM.
intracellular signalling pathway, indicated by elevated
Dab1 and ERK1/2 phosphorylation in primary cortical
neurons (Figure 2a–h). Moreover, we observed an
increase in phosphorylated AKT to total AKT
(Figure 2i–l) and a decrease in active GSK-3β via phos-
phorylation (Figure 2m–p). In neurons, GSK-3β regulates
various processes critical in functional synaptic plasticity,
highlighting GSK-3β as a key signalling molecule in nor-
mal brain functioning (Jaworski et al., 2019). Our results
suggest that R36 is consistent with full-length Reelin and
R3456 in activating this signalling pathway (Li
et al., 2023). To further explore the functional efficacy of
the R36 fragment, we performed a single ICV injection of
the R36 protein in the HRM and WT mice. As previously
shown in our study with R3456 supplementation (Li
et al., 2023), HRM control exhibited a deficiency in loco-
motor activity during open field, indicated by a decrease
in total distance travelled when comparing HRM PBS to
WT animals (Figure 3b). We did not evidence any differ-
ences between groups on anxiety-like behaviour, as all
the treatment groups spend the same amount of time in
the centre (Figure 3c). In addition, heterozygous reeler
mice displayed a deficit in spatial learning as shown as
decreased time spent in quadrant (Figure 3d) and
decreased target platform crossings (Figure 3e) during
Morris water maze. Finally, HRM exhibited a decrease in
associative learning, evidenced by a decrease in time
freezing when compared with WT animals during con-
textual but not cued fear conditioning (Figure 3h,i). Our

MORRILL ET AL.
results on spatial learning during Morris water maze
were consistent with another model of Reelin deficiency
(Paylor et al., 1999). Nonetheless, our data on locomotor
activity and spatial learning deficiencies in the HRM are
not in accordance with studies from other teams, which
did not find any difference between wildtypes animals
and HRM in total distance travelled during open field or
learning during Morris water maze (Qiu et al., 2006;
Teixeira et al., 2011). This difference might be attributed
to the age of the animals, which were much younger than
in the experiment described here (6 weeks in Qiu
et al., 2006, and 8 weeks in Teixeira et al., 2011, vs. 12–
14 weeks old in the current experiment). However, there
seem to be a consensus on associative learning deficiency.
Deficit in contextual fear conditioning, but not cued, in
HRM control mice when compared with wild type was
consistent with other reports, even in the younger mice
(Qiu et al., 2006; Rogers et al., 2013; Weeber et al., 2002).
We demonstrate that a single administration of the R36
protein in the HRM was able to recover behavioural defi-
cits in the open field maze (Figure 3b), the hidden plat-
form water maze (Figure 3d,e) and associative fear
conditioning (Figure 3h). This supports the use of R36,
essentially as an analogue of Reelin, for therapeutically
increasing Reelin signalling in vivo. Our results are con-
sistent with previous work on full length Reelin single
injection and R3456 single injection in this mouse model
(Li et al., 2023; Qiu et al., 2006; Weeber et al., 2002).
Support for Reelin therapies is growing as more
groups investigate the effects of Reelin supplementa-
tion and enhancement as recently outlined in a review
by Tsuneura et al. (2022). Treatment with Reelin has
been demonstrated to increase dendritic spine density
in primary cultured hippocampal neurons (Kim
et al., 2015). Others have reported benefits of Reelin
supplementation in a mouse model of Angelman syn-
drome and in WT mice (Rogers et al., 2011, 2013) as
well as in a model of maternal induced activation of
schizophrenia (Ibi et al., 2020). Additionally, our labo-
ratory has reported that administration of R3456 into
HRM and a mouse model of fragile X syndrome
induced recovery of known cognitive deficits (Li
et al., 2023; Morrill et al., 2022).
In the literature, exogenous supplementation with
Reelin has been linked to increased dendritic spine den-
sity in primary hippocampal neurons and increased
puncta numbers of synaptophysin and PSD95. It has been
proposed that the Ca2+/calmodulin-dependent protein
kinase II β subunit may be necessary for these effects of
exogenous Reelin supplementation on dendritic spine
density (Kim et al., 2015). Previous groups have demon-
strated a baseline reduction in dendritic spine density in
hippocampal pyramidal neurons of heterozygous and
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1667
homozygous reeler mice. In hippocampal cultures, this
reduction was restored with treatment of full-length
Reelin (Niu et al., 2008). To better understand the results
we observed with treatment of R36 protein, we chose to
examine PSD-95 and synaptophysin, markers of post-
and pre-synaptic proteins, respectively. We demon-
strated that HRM contain a baseline reduction in levels
of post-synaptic protein-PSD95 and following supple-
mentation of R36 protein, we were able to recover the
levels of PSD95 in HRM (Figure 4c,d). For synaptophy-
sin, we did not observe a baseline reduction or any
change in levels of protein between any of the groups
(Figure 4a,b). The results we observed in pre- and post-
synaptic markers with Reelin supplementation may
indicate an improvement in dendritic spine maturation,
but further work needs to be done to better visualize
and quantify any effects on dendritic spine density and
maturation. It has been shown previously that exoge-
nous Reelin supplementation may increase the number
of puncta and overall dendritic spine density, future
work could include Golgi staining to better visualize
spine dynamics and localization of pre- and post-
synaptic markers.
In summary, we show that a novel construct encom-
passing the Reelin EGF-like repeats 3 and 6 can elicit
receptor dimerization and downstream signalling. We
demonstrate that a single injection of this R36 protein
directly into the brains of HRM rescues spatial learning
and memory deficits and associative learning deficits. We
believe that this supports the potential use of R36 as a
therapeutic approach for the treatment of diseases which
exhibit reduced Reelin signalling. Additionally, we
acknowledge that when considering the practicality of
Reelin supplementation as a therapeutic, the use of a pro-
tein therapy such as R36 injection, is likely impractical as
this would involve repeated cerebral spinal fluid (CSF)
injections. Increasingly, gene therapy approaches using
adeno-associated virus (AAV)-based vectors are showing
promise in offering therapeutic treatment to a broad
array of diseases (Orlowski et al., 2020). A gene therapy
approach would offer a longer-term application. Future
work should consider exploring AAV constructs to better
understand the plausibility and effectiveness of R36 as a
potential long-term therapeutic approach.
AUTHOR CONTRIBUTIONS
Kevin R. Nash and Edwin J. Weeber designed research;
Nicole K. Morrill and Qingyou Li conducted research;
Kevin R. Nash, Nicole K. Morrill and Aurelie Joly-Amado
analysed data, performed statistical analysis and wrote
the paper. Kevin R. Nash and Edwin J. Weeber had pri-
mary responsibility for final content. All authors read
and approved the final manuscript.

1668
ACK NO WLE DGE MEN TS
This research did not receive any specific grant from
funding agencies in the public, commercial or not-for-
profit sectors.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
DATA AVAILABILITY STATEMENT
Data described in the manuscript, code book and analytic
code will be made available upon request pending
(e.g., application and approval, payment and other).
P EE R R EV IE W
The peer review history for this article is available at
https://www.webofscience.com/api/gateway/wos/peer-
review/10.1111/ejn.15971.
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How to cite this article: Morrill, N. K., Li, Q.,
Joly-Amado, A., Weeber, E. J., & Nash, K. R.
(2023). A novel Reelin construct, R36, recovered
behavioural deficits in the heterozygous reeler
mouse. European Journal of Neuroscience, 57(10),
1657–1670. https://doi.org/10.1111/ejn.15971