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Reelin reverts biochemical, physiological and cognitive alterations in mouse models of Tauopathy

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Reelin is an extracellular protein crucial for adult brain plasticity. Moreover, Reelin is protective against amyloid-β (Aβ) pathology in Alzheimer's Disease (AD), reducing plaque deposition, synaptic loss and cognitive decline. Given that Tau protein plays a key role in AD pathogenesis, and that the Reelin pathway modulates Tau phosphorylation, here we explored the involvement of Reelin in AD-related Tau pathology. We found that Reelin overexpression modulates the levels of Tau phosphorylation in AD-related epitopes in VLW mice expressing human mutant Tau. In vitro, Reelin reduced the Aβ-induced missorting of axonal Tau and neurofilament proteins to dendrites. Reelin also reverted in vivo the toxic somatodendritic localization of phosphorylated Tau. Finally, overexpression of Reelin in VLW mice improved long-term potentiation and long-term memory cognitive performance thus masking the cognitive and physiological deficits in VLW mice. These data suggest that the Reelin pathway, which is also protective against Aβ pathology, modulates fundamental traits of Tau pathology, strengthening the potential of Reelin as a therapeutic target in AD.
Reelin overexpression rescues cognitive deficits in VLW model. a) Open field. Quantification of the mean distance travelled by mice (in m) in either X, Y and Z axes inside the test cage (upper panel). The time (in min) that animals spent in the central area and in the periphery are also represented (lower panel). Data are represented as mean ± SEM; no significant differences were observed between groups (one-way ANOVA). n = 9-19 animals per genotype. b) Accelerating rotarod. Bars show the mean number of falls (left) and the time (in s; right) that the animals remained on the rotating bar over the two successive trials. Data are represented as mean ± SEM; no significant differences were observed between the four experimental groups (one-way ANOVA). n = 10-19 animals per genotype. c) Elevated path test. The bars indicated the mean latency (in s) to fall of the four experimental groups during the two trials of the test. Data are represented as mean ± SEM; no significant differences were observed between groups (two-way ANOVA). n = 10-14 animals per genotype. d) Passive avoidance test. Latency to enter the dark compartment for the control, TgRln, VLW, and TgRln/VLW groups was measured to a maximum of 180 s. Data are represented in cumulative frequencies; significant differences between genotypes was determined by curve comparisons in pairs using Log-Rank (Mantel Cox) Test for each time point of analysis¸*p < 0.05; **p < 0.01. n = 9-19 animals per genotype. Although the latency between the four groups did not differ during the acquisition session, the latency of the VLW group during the retention session was smaller than for the other three groups. Indeed, the cumulative frequency presented by the VLW group for the retention test (24 h) was significantly smaller than for the other three groups.
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Progress in Neurobiology
journal homepage: www.elsevier.com/locate/pneurobio
Reelin reverts biochemical, physiological and cognitive alterations in mouse
models of Tauopathy
Daniela Rossi
a,b,c
, Agnès Gruart
d
, Gerardo Contreras-Murillo
d
, Ashraf Muhaisen
a,b,c
, Jesús Ávila
e
,
José María Delgado-García
d
, Lluís Pujadas
a,b,c,
*
,1
, Eduardo Soriano
a,b,c,f,
*
,1
a
Vall dHebron Institut de Recerca, 08035, Barcelona, Spain
b
Department of Cell Biology, Physiology and Immunology, and Institute of Neurosciences, University of Barcelona, 08028, Barcelona, Spain
c
Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031, Madrid, Spain
d
Division of Neurosciences, Universidad Pablo de Olavide, 41013, Sevilla, Spain
e
Centro de Biología Molecular Severo Ochoa, 28049, Madrid, Spain
f
ICREA Academia, 08010, Barcelona, Spain
ARTICLE INFO
Keywords:
Intracellular signaling
Reelin
Tauopathy
Electrophysiology
Cognition
Alzheimers disease
ABSTRACT
Reelin is an extracellular protein crucial for adult brain plasticity. Moreover, Reelin is protective against amy-
loid-β(Aβ) pathology in Alzheimers Disease (AD), reducing plaque deposition, synaptic loss and cognitive
decline. Given that Tau protein plays a key role in AD pathogenesis, and that the Reelin pathway modulates Tau
phosphorylation, here we explored the involvement of Reelin in AD-related Tau pathology. We found that Reelin
overexpression modulates the levels of Tau phosphorylation in AD-related epitopes in VLW mice expressing
human mutant Tau. in vitro, Reelin reduced the Aβ-induced missorting of axonal Tau and neurolament proteins
to dendrites. Reelin also reverted in vivo the toxic somatodendritic localization of phosphorylated Tau. Finally,
overexpression of Reelin in VLW mice improved long-term potentiation and long-term memory cognitive per-
formance thus masking the cognitive and physiological decits in VLW mice. These data suggest that the Reelin
pathway, which is also protective against Aβpathology, modulates fundamental traits of Tau pathology,
strengthening the potential of Reelin as a therapeutic target in AD.
1. Introduction
Reelin is an extracellular matrix protein that controls neuronal
migration in the embryonic brain, being a major actor in corticogenesis
and in the lamination of other brain areas, such as the cerebellum
(DArcangelo and Reeler, 1998;Rice and Curran, 2001;Soriano and Del
Rio, 2005). Reelin is also expressed in the adult brain (Alcantara et al.,
1998), where it regulates the induction of synaptic plasticity (Herz and
Chen, 2006) and potentiates glutamatergic neurotransmission, directly
regulating N-methyl-D-aspartate (NMDA) receptor subunit tracking
and function (Durakoglugil et al., 2009;Groc et al., 2007). The over-
expression of Reelin in the adult mouse brain (TgRln model) drives an
increase in excitatory synaptic contacts, hypertrophy of dendritic
spines, and enhanced long-term potentiation (LTP) responses (Pujadas
et al., 2010). These observations thus indicate that Reelin is potentially
involved in the modulation of cognition, learning and memory.
The homeostasis of key players underlying synaptic plasticity is
fundamental for the maintenance of cognitive functions in the adult
brain and for implications in neurodegenerative diseases. Alzheimers
disease (AD), the most prevalent form of dementia worldwide (Reitz
and Mayeux, 2014), is characterized by abnormal dendritic spine
morphology and density (Spires-Jones and Knafo, 2012) and by sy-
naptic dysfunction (Sivanesan et al., 2013). Indeed, synaptic loss shows
a greater correlation with cognitive impairment than histopathological
hallmarks of the disease such as amyloid plaques, thus leading to
consider AD as a synaptic failure-disease (Selkoe, 2002). Of note,
both in the J20 AD mouse model (carrying the Swedish and the Indiana
mutations of the human Amyloid Precursor Protein) and in AD patients,
depletion of Reelin has been reported as one of the early events in this
pathology (Herring et al., 2012;Chin et al., 2007). Consistently, over-
expression of Reelin in the J20 AD model reduces not only amyloid
plaque deposition but also synaptic loss, while rescuing memory-
https://doi.org/10.1016/j.pneurobio.2019.101743
Received 19 December 2018; Received in revised form 24 October 2019; Accepted 18 December 2019
Corresponding authors at: Department of Cell Biology, Physiology and Immunology, and Institute of Neurosciences, University of Barcelona, 08028, Barcelona,
Spain.
E-mail addresses: lluis.pujadas@ub.edu (L. Pujadas), esoriano@ub.edu (E. Soriano).
1
LP and ES are co-senior authors.
Progress in Neurobiology 186 (2020) 101743
Available online 20 December 2019
0301-0082/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
T
associated cognitive impairment (Pujadas et al., 2014).
Although Aβoligomers have been widely recognized as the main
species responsible for synaptic dysfunction in AD (Selkoe, 2008;Walsh
et al., 2002;Haass and Selkoe, 2007), recent data indicate that their
toxicity is mediated by Tau protein (Ittner and Gotz, 2011;Ittner et al.,
2010;Bloom, 2014), whose intracellular aggregates, known as neuro-
brillary tangles (NFTs), are another hallmark of the disease. In addi-
tion, Tau pathology in the human brain has a high correlation with
brain atrophy and severe cognitive impairment (Jagust, 2018).Together
with MAP1 and MAP2, Tau is one of the major brain-specic micro-
tubule-associated proteins (MAPs) involved in the stabilization of the
microtubule network, among other physiological functions (Wang and
Mandelkow, 2016). Tau develops its brain-specic functions as an ax-
onal protein, and its activity is regulated by its degree of phosphor-
ylation at dierent tyrosine, threonine or serine residues. In the AD
brain, phosphorylated Tau (phospho-Tau) levels are three- to four-fold
higher than in the healthy brain, and Tau starts to be phosphorylated at
abnormal sites (Kopke et al., 1993;Iqbal et al., 2010). In this hyper-
phosphorylatedstate, Tau detaches from microtubules and mis-
localizes to the somatodendritic compartment, where it polymerizes
into paired helical laments (PHFs), nally resulting in the formation of
NFTs (Wang and Mandelkow, 2016;Ballatore et al., 2007). Moreover,
when abnormally modied, Tau becomes enriched in dendritic spines
and thus interferes with neurotransmission (Hoover et al., 2010). Aβ
oligomers promote both Tau hyperphosphorylation and its mis-
localization to dendrites and postsynaptic enrichment, nally causing
depletion of dendritic spines (Zempel et al., 2013;Zempel et al., 2010).
In the context of AD pathology, in addition to interacting with and
reducing the toxicity of Aβspecies (Pujadas et al., 2014), Reelin sig-
naling triggers an intracellular cascade in which AKT kinase is induced.
This activation nally leads to the modulation of the activity of GSK-3β,
the major kinase for Tau protein (Gonzalez-Billault et al., 2005;Beert
et al., 2002). Indeed, mutant mice decient in Reelin, in its transducer
Dab1, or in Reelin receptors ApoER2 and/or VLDLR show increased
levels of Tau phosphorylation (Hiesberger et al., 1999;Ohkubo et al.,
2003). Of note, the APOE4 protein, which binds APOER2 receptors, has
recently been found to exacerbate Tau pathology (Shi et al., 2017).
Moreover, Reelin haploinsuciency (heterozygous reeler)inAD
mice results in accelerated AD-like pathology, enhancing both amyloid
plaque burden and NFT deposition (Kocherhans et al., 2010), while
adult conditional Reelin knock-out mice display severe memory im-
pairment when exposed to very low amounts of amyloid deposition
(Lane-Donovan et al., 2015). Conversely, whether the overactivation of
Reelin signaling is able to reverse Tau hyperphosphorylation in the
context of AD has not been explored to date.
Here we examined whether transgenic Reelin overexpression in an
AD background alters pathological levels of phospho-Tau and amelio-
rates the cognitive decits associated with Tau pathology. We show
that Reelin overexpression modulates in vivo the levels of Tau phos-
phorylation at some AD-related epitopes in the VLW Tauopathy mouse
model (Lim et al., 2001). Concomitantly, both in vitro and in vivo, Reelin
reduced the toxicity-associated somatodendritic localization of NF and
phospho-Tau. Finally, VLW mice showed improved LTP responses and
cognitive performance in the passive avoidance paradigm upon Reelin
overexpression. Taken together, our results complement the previously
reported benecial eect of Reelin in AD pathology and confer the
Reelin pathway a pivotal role as a negative regulator of AD progression,
by antagonizing both Aβand Tau pathologies.
2. Material and methods
2.1. Animals
The TgRln (Tg1/Tg2; pCamKII-tTA/tetO-rlM) mouse is a condi-
tional regulated double transgenic line based on a Tet-oregulated
binary system that achieves Reelin overexpression under the control of
calcium/calmodulin-dependent kinase II αpromoter (pCaMKIIα)
(Pujadas et al., 2010). The GSK-3β(Tg1/Tg3; pCaMKII-tTA/BitetO β-
Gal/GSK-3β) mouse is a conditional regulated double transgenic line
showing GSK-3βprotein overexpression (and the reporter gene β-Gal)
also under the control of pCaMKIIα(Lucas et al., 2001). The VLW (Tg4;
pThy1-Tau-VLW) mouse is a constitutive transgenic line expressing
human Tau protein with four tubulin-binding repeats (increased by
FTDP-17 splice donor mutations) and three FTDP-17 missense muta-
tions: G272 V, P301 L, and R406W under the control of thy1 promoter
gene cassette (Lim et al., 2001). TgRln/GSK-3β(Tg1/Tg2/Tg3) and
TgRln/VLW (Tg1/Tg2/Tg4) mice are triple transgenic, obtained by
interbreeding the previously described strains. Control animals used
throughout the study were littermates of transgenics not bearing
overexpression of Reelin, GSK-3β, or VLW (i.e. wild-types, single car-
riers of Tg1, Tg2 or Tg3 transgenes, or double Tg2/Tg3). All the
transgenic animals used in this study are kept in homozygosis for each
transgene. Both male and female animals were used, thus representing
the diversity of the population in biochemical and histological experi-
ments; only males were used in behavioral and electrophysiological
experiments due to the well-known variability in such tests. Mice were
bred, studied and processed in the animal research facility at the Fa-
culty of Pharmacy of the University of Barcelona or sent to the animal
research facility of the Pablo de Olavide University for behavioral
studies. Animals were provided with food and water ad libitum and
maintained in a temperature-controlled environment in a 12/12 h light-
dark cycle. All the experiments involving animals were performed in
accordance with the European Community Council directive and the
National Institute of Health guidelines for the care and use of laboratory
animals. Experiments were also approved by the local ethical commit-
tees.
2.2. Chemicals
Diaminobenzidine reagent (DAB), Hydrogen Peroxide (H
2
O
2
), 4-6-
Diamino-2-Phenylindole (DAPI), Triton X-100, Dimethyl Sulfoxide
(DMSO), Nissl (Cresyl violet acetate and Thionine acetate salt),
Hexadeuterodimethyl sulfoxide (DMS=-d6), magnesium chloride,
HEPES buer, EGTA, 1,1,1,3,3,3-Hexauoro-2-propanol and
Phalloidin-Tetramethylrhodamine B isothiocyanate (red-phalloidin)
were from Sigma. Paraformaldehyde (PF), Eukitt mounting medium,
Glycerol, Ethylene Glycol, Phosphate Bue TRIS buer, sodium
chloride, sodium orthovanadate, sodium uoride and gelatine were
from Panreac. Mowiol 488 reagent was from Calbiochem. Nickel
ammonium sulfate was from Analyticals. Tetra-sodium pyrophosphate
was from Fluka. Glycerol was from Merck. Protein A and Protein G
Sepharose 4 Fast Flow were from GE Healthcare.
2.3. Antibodies
Mouse anti-phopsho-Tau (ser 396/404) PHF-1 was a kind gift from
Dr. Peter Davies, Albert Einstein College of Medicine, New York, NY
(Greenberg et al., 1992). The following commercial primary antibodies
were used: anti-Reelin (clone G10) (Chemicon); anti-phospho-Tau
(ser202/thr205, clone AT8, Innogenetics); anti-total Tau (clone Tau-5,
Millipore); anti-total Tau K9JA (DAKO, A0024); anti-actin (Chemicon,
MAB1501); anti-phospho-Tau T205 (ThermoFisher, 44-738G); anti-
neurolament (NF200, Sigma, N4142); anti-calbindin (Swant, CB38);
anti-MAP2 (Sigma, M1406); anti-human Tau HT7 (Thermosher,
MN1000); and anti-βGalactosidase (AB986, Millipore). The HRP-la-
beled secondary antibodies used for western blot were from DAKO.
Biotinylated-secondary antibodies and Streptavidin-biotinylated/HRP
complex were from GE Healthcare. F(ab')2 fragment anti-mouse IgG
was supplied by Jackson Immuno Research. The uorescent secondary
antibodies used for immunouorescence were purchased from In-
vitrogen (Alexauor).
D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
2
2.4. Software
NDP.view2 (Hamamatsu Photonics) was used to magnify areas of
interest from Nanozoomer-scanned immunohistochemical slides. Image
J was used as a general tool for the analysis, measuring, processing and
quantication of images. Gel-Pro Analyzer was used for the quanti-
cation of WB-scanned lms. GraphPad Prism was used for graphical
representation and statistical analysis of the data.
2.5. Aβ-derived diusible ligands (ADDLs) preparation
ADDLs were prepared as described (Pujadas et al., 2014;Lambert
et al., 2001). Briey, Aβ
42
was dissolved in hexauoro-2-propanol
(1 mg/ml) and aliquoted in low-binding Eppendorf tubes. Hexauoro-
2-propanol was then removed by freeze-drying. An aliquot of Aβ
42
was
dissolved in anhydrous DMSO-d6 to 5 mM and further diluted with ice-
cold F12 medium (GIBCO) without phenol red to 100 mm. This solution
was incubated at 4 °C for 24 h and then centrifuged at 14000gfor
10 min. The Aβ
42
concentration in the supernatant was determined
using the Bradford assay and found to range between 60 and 100 mM.
The peptide solution was diluted to the desired concentrations for pri-
mary hippocampal cell treatments (1 and 3 μM).
2.6. Primary hippocampal cultures
Hippocampal neurons were obtained from E16 CD1 mouse embryos
(animal research facility at the Faculty of Pharmacy of the University of
Barcelona). Brains were dissected in PBS containing 0.6 % glucose, and
hippocampi were excised. After trypsin (GIBCO) and DNAse (Roche
Diagnostics) treatments, hippocampi were dissociated by gentle
sweeping. Cells were counted and seeded at 3 × 10
4
cells per well in
four-well plates onto poly-D-lysine-coated coverslips in Neurobasal
medium containing B27 supplement (GIBCO) supplemented with
GlutaMAX (GIBCO), Penicillin-Streptomicin (GIBCO) and 1:5 of con-
ditioned media obtained from astrocyte cultures. Neuronal cultures
were kept for 21 days in vitro (DiV) in 5 % CO2 at 37 °C before treat-
ment. Cells were incubated for 3 h at 37 °C with ADDLs (1 and 3 μM) or
corresponding volumes of vehicle (0.1 % DMSO in F12 medium), with
or without the addition of 5 μg/ml Reelin (or equivalent volumes of
Mock). Addition of vehicle/ADDLs and Mock/Reelin was made se-
quentially during treatments, without mixture of the two agents outside
the culture media. Cells were then washed 3 times with phosphate
buer saline (PBS) and xed for 20 min with 4 % paraformaldehyde
(PF) in 0.1 M phosphate buer (PB). For immunouorescence, cells
were blocked for 2 h at room temperature (RT) with PBS containing 10
% of normal goat serum (NGS) and 0.1 % Triton in 0.2 % of gelatin.
Primary antibodies (NF200 1:1000; K9JA 1:50000; MAP2 1:500) were
incubated overnight at 4 °C. Incubation with uorescent secondary
antibody (1:200; 2 h at RT), red phalloidin (2 μg/mL) and DAPI
(0.02 μg/mL) was performed in PBS-5 % NGS. Coverslips were mounted
in Mowiol.
2.7. Fluorescence imaging and detection of missorting
Hippocampal neurons were observed with a 40X objective on an
Olympus CellR/ScanR microscope. Micrograph mosaics of 10 × 10
images were randomly taken from coverslips for each condition. Fields
covering an area of 2.9-3 mm
2
and containing a range from 30 to 300
neurons were quantied. Tau and NF200 missorting was determined by
counting the number of neurons displaying at least one dendrite with
increased signal versus the total number of neurons per mosaic/eld in a
semi-automated manner when possible. NF200 missorting quantica-
tion was performed by implementing a macro for automate detection of
candidate-neurons with missorted signal; briey, cells in the culture
were detected using cell counting upon nuclei marker (blue, DAPI
staining), then compared using a colocalization mask with neuronal
marker (red, MAP2 staining) to identify neurons and again compared
using a colocalization mask with abnormal axonal somatic staining
(green, NF200 staining) to identify candidate-neurons with missorted
signal; those candidate cells were nally conrmed by individual ob-
servation in double blind experiment to determine neurons with mis-
sorted NF200. Tau missorting quantication was performed by
counting in each eld the total number of neurons and those with
missorted phenotype in double blind experiments; neurons in the cul-
ture were individually identied considering cell morphology (red-
Phalloidin staining) and Tau content (green, Tau K9JA staining) and
neurons with missorted signal were individually identied considering
Tau distribution inside the cell.
Images obtained from ScanR microscopy were quantied along the
proximal 20 μm of primary dendrites in both translocated and un-
translocated dendrites. Intensities for NF200- and Tau-signaling were
determined in all conditions by performing a plot-prole along trans-
located dendrites from cell soma in 2 μm thickness. Untreated un-
translocated dendrite intensities were used in each experiment for
normalization. Quantitative data was obtained from 3 to 4 independent
experiments (each including all treatment conditions). Each experiment
included 23 replicates per condition. Each independent experiment
was processed and quantied in bulk.
2.8. Western blot
To obtain protein extracts from brain tissues, animals were killed by
dislocation and brains were immediately removed. Specic brain re-
gions were rapidly dissected, frozen in liquid nitrogen, and stored at
80 °C before processing. Frozen pieces of tissues were also lysed using
power homogenizer (Polytron) in 10 volumes of lysis buer (50 mM
HEPES, pH 7.5, 150 mM sodium chloride, 1.5 mM magnesium chloride,
1 mM EGTA, 10 % glycerol, and 1 % Triton X-100) containing Complete
Mini protease inhibitor cocktail (Roche) and phosphatase inhibitors
(10 mM tetra-sodium pyrophosphate, 200 μM sodium orthovanadate,
and 10 mM sodium uoride).
Samples were sonicated, insoluble debris was removed by cen-
trifugation (15 min, 16,000 X g), and supernatants were stored at
-80 °C. A preclearing step was included when needed for removal of
endogenous immunoglobulins by incubating samples with 30 μLof
Protein-G beads for 90 min at 4°C then recovering cleaned supernatants
by centrifugation. Samples were diluted 1:6 with 6X loading buer
(0.5 M Tris-HCl, pH 6.8, 2.15 M β-mercaptoethanol, 10 % SDS, 30 %
glycerol, and 0.0.2 % bromophenol blue), boiled for 3 min at 95 °C.
Samples were resolved by SDS-8 % polyacrylamide gels and transferred
onto nitrocellulose membranes. Membranes were then blocked for 1 h
at RT in TBST (Tris 10 mM pH 7.4, sodium chloride 140 mM (TBS) with
0.1 % Tween 20) containing 5 % non-fat milk or 3 % BSA. Primary
antibodies were incubated for 90 min in TBST-0.02 % azide (anti-actin
1:100,000; PHF-1 1:500; AT8 1:500; T205 1:1000; total Tau K9JA
1:20,000; or total Tau5 1:1000). After incubation with anti-mouse or
anti-rabbit secondary HRP-labeled antibodies for 1 h at RT (diluted
1:2000 in TBST-5 % non-fat milk), membranes were developed with the
ECL system (GE Healthcare).
2.9. Histology
Animals were anesthetized and perfused for 20 min with 0.1 M PB
containing 4 % of PF. Brains were removed, postxed overnight with
PB-4 % PF, cryoprotected with PB-30 % sucrose and frozen. Brains were
sectioned coronally at 30 μm in a cryostat and maintained at -20 °C in
PB-30 % glycerol-30 % ethylene glycol. For immunodetection of anti-
gens, sections were incubated with 10 % methanol-3 % H
2
O
2
and then
blocked for 2 h at RT with PBS containing 10 % of either normal goat
serum (NGS) or normal horse serum (NHS), 0.2 % of gelatine, 0.3 %
Triton and F(ab')2 fragment anti-mouse IgG (1:300) when needed.
Primary antibodies (PHF-1 1:150; T205 1:300; calbindin 1:3000; HT7
D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
3
1:500; anti-βGalactosidase 1:200; anti-Reelin 1:200) were incubated
overnight at 4 °C with PBS-5 % NGS or 5 % NHS. For im-
munohistochemistry, sequential incubation with biotinylated sec-
ondary antibody (1:200; 2 h at RT) and streptavidin-HRP (1:400; 2 h at
RT) was performed in PBS-5 % NGS or 5 % NHS. Bound antibodies
were visualized by reaction using DAB and H
2
O
2
as peroxidase sub-
strates, adding nickel ammonium sulfate in the solution in some cases.
Sections were dehydrated, and mounted (Eukitt). For each im-
munostaining, we developed in bulk the tissue sections from the dif-
ferent genotypes, for all the steps of the process including processing,
staining, imaging and analysis. To evaluate the immunohistochemical
phospho-Tau signal we quantied the area occupied with positive
staining in each of the areas of interest (i.e. CA1 stratum radiatum and
granular layer for the VLW strain; and CA3 stratum lucidum for the
GSK-3βstrain). Images obtained from Nanozoomer scans at 20X were
enlarged to the required magnication with NDPview2 software and
processed with ImageJ in a double-blind analysis as follows: rst, the
area to be analyzed was delimited on 8-bit converted images; second,
thresholds of positive/negative signal were determined in areas without
staining in the same preparations; third, thresholds were applied to
generate binary images; nally, the percentage of the area of interest
occupied with positive signal was calculated on binary images.
2.10. Behavioral analysis
2.10.1. Open eld
The open eld test is a behavioral motor task aimed at determining
the general locomotor activity of experimental animals, their will-
ingness to explore new environments and their anxiety levels.
Experiments were carried out in an open eld (a box of
28 × 28 × 21 cm) apparatus (Actifot 809 from Cibertec S.A., Madrid,
Spain). Mice (control = 10; TgRln = 8; VLW = 15; TgRln/VLW = 10)
were placed in the center of the arena and observed for 15 min. The
apparatus was provided with infrared lights, located every 2 cm, in the
three (X, Y, Z) spatial axes. Animalsmovements in the arena were
quantied automatically with the help of a computer program
(MUX_XYZ16 L), also from Cibertec S.A. The computer program also
allowed us to discriminate when the animal was located at the center
(17 × 17 cm) or at the periphery (the surrounding area) of the arena.
The apparatus was located in a sound-proof room and the experimental
area was homogeneously dimly illuminated. The whole apparatus was
carefully cleaned with alcohol (70° proof) after each use. Total mobility
in the X, Y, and Y (i.e., rearing) axes was quantied in arbitrary units, as
was the total time (in min) spent in the center and the periphery of the
arena.
2.10.2. Elevated path test
This test was designed to check the motor ability of the animals. The
elevated path consisted of a 40 cm long, 5 cm wide bar located 60 cm
over a soft cushion. Each mouse (control = 19; TgRln = 13; VLW = 10;
TgRln/VLW = 14) was placed in the center of the elevated bar and
allowed a maximum of 30 s to reach one of the platforms (12 × 12 cm)
located at each end of the bar. We quantied latency to fall (in s) (test
1). The test was repeated 24 h later (test 2).
2.10.3. Rotarod
The rotarod test is a behavioral task that assesses motor coordina-
tion performance. In this study, we used an accelerating rotarod
treadmill (Ugo Basile, Varese, Italy). Mice (control = 13; TgRln = 9;
VLW = 10; TgRln/VLW = 14) were placed on the rod and tested at
220 rpm (of increasing speed) the rst day, for a maximum of 300 s at
each speed. Between trials, mice were allowed to recover in their cages.
The total time that each animal remained on the rod was computed as
latency to fall, recorded automatically by a trip switch under the oor
of each rotating drum. Mice were tested two times with an interval of
one hour during the same session. Animals were re-tested 24 and 72 h
later. Results were evaluated by averaging the data from the 4 trials
corresponding to sessions 2 and 3.
2.10.4. Passive avoidance test
In this case, mice (control = 19; TgRln = 13; VLW = 9; TgRln/
VLW = 14) were placed in darkness for 5 min before training. They
were then placed individually in an illuminated box (10 × 13 × 15 cm)
connected to a dark box of the same size. The dark box was equipped
with an electric grid oor and separated by an automatic door (passive
avoidance device, Ugo Basile, Comerio, VA, Italy). This door was
opened 60 s later. Entry of animals into the dark box was punished by a
timed electric foot-shock (0.5 mA, 1 s). After 24 h, pre-trained animals
were placed in the illuminated box again and observed for 3 min. The
time that the mice took to enter the dark box was noted, and the mean
time was calculated for each experimental group. Animals were re-
tested 24 h later. Latency (in s) was determined automatically by the
experimental device (Eleore et al., 2007).
2.11. Electrophysiological recordings
Animals were anesthetized with 0.81.5 % isouorane (Astra
Zeneca, Madrid, Spain) delivered from a calibrated mask (Cibertec,
Madrid, Spain). Animals were chronically implanted with bipolar sti-
mulating electrodes in the right (contralateral) Schaer collater-
alcommissural pathway of the dorsal hippocampus (2 mm lateral and
1.5 mm posterior to bregma, and 1.01.5 mm below brain surface;
Paxinos and Franklin, 2004) and with a recording electrode in the ip-
silateral stratum radiatum underneath the CA1 area (1.2 mm lateral and
2.2 mm posterior to bregma, and 1.01.5 mm below brain surface).
Electrodes were made of 50 m, Teon-coated tungsten wire (Advent
Research Materials, Eynsham, UK). The nal location of the recording
electrode in was determined following the eld potential depth prole
evoked in the CA1 area by paired (40-ms interval) pulses presented to
the ipsilateral Schaer collateral pathway (Gruart et al., 2006). The
recording electrode was xed at the site where a reliable monosynaptic
fEPSP was recorded. Two bare silver wires (0.1 mm in diamater) were
axed to the skull as ground. Electrodes were connected to a 6-pin
socket (RS-Amidata, Madrid, Spain). The socket was xed to the skull
with the help of three small screws and dental cement. Further details
of this chronic preparation has been described elsewere (Gruart et al.,
2006). Animals were allowed a week to recover before the start of the
recording sessions.
Electrophysiological recordings were carried out in alert behaving
animals placed in ventialted small (6 × 6 × 6 cm) containers.
Recordings were carried out with the help of high-impedance probes
(2 × 10
12
Ω, 10 pF) connected to dierential ampliers within a
bandwidth of 0.110 kHz (P511, Grass-Telefactor, West Warwick, RI,
USA). Electrical stimulations were provided across isolation units con-
nected to a CS-220 stimulator (Ciberted, Madrid, Spain).
For input/output curves, monosynaptic fEPSPs were evoked in the
CA1 area by single (100 μs, square, and negative-positive) pulses ap-
plied to Schaer collaterals. These pulses were presented at increasing
intensities ranging from 20 μA to 400 μA, in steps of 20 μA. In order to
avoid interactions with the preceding stimuli, an interval of 30 s was
allowed between each pair of pulses (Madronal et al., 2007).
For the paired-pulse facilitation at the CA3-CA1 synapse, we used
the same type of pulses indicated above, but presented in pairs at in-
creasing inter-pulse intervals (10, 20, 40, 100, 200 and 500 ms). For
each animal, the stimulus intensity was set at 3040 % of the intensity
necessary for evoking a maximum fEPSP response (Gruart et al., 2006;
Gureviciene et al., 2004). An additional criterion for selecting stimulus
intensity was that a second stimulus, presented 40 ms after a con-
ditioning pulse, evoked a larger (20 %) synaptic eld potential (Bliss
and Gardner-Medwin, 1973). Intervals between pairs of pulses were set
at 30 s, to avoid unwanted interactions evoked by pre- or post-synaptic
mechanisms.
D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
4
For evoking LTP, we used a high-frequency stimulation (HFS) train
consisting of ve 200 Hz, 100 ms trains of pulses at a rate of 1/s. This
protocol was presented six times, at intervals of 1 min. As indicated
above for functional synaptic plasticity, pulse intensity (50400 A) was
set at 3040% of the amount required to evoke a maximum fEPSP re-
sponse for baseline recordings (15 min) and after the HFS train
(60 min). Recording sessions were repeated for 4 additional days
(30 min each) To avoid evoking a population spike and/or unwanted
electroencephalographic (EEG) seizures, the stimulus intensity during
the HFS train was set at the same amount as that used for generating
baseline recordings.
2.12. Statistical analysis
Statistical analysis was performed using GraphPad Prism software.
Outlier values were identied by applying the ESD method (extreme
studentized deviate) and excluded from the analysis.
For quantication of WB in Fig. 1 (n = 34 animals per genotype),
densitometric analysis of protein bands of interest was performed using
GelPro software and normalized for loading controls (i.e. actin). To
include data obtained from dierent gels, a specic sample was loaded
in each gel and used for normalization. Signicance between the two
genotypes expressing human Tau was analyzed by using the unpaired
Student's t-test
To determine Tau- and NF200-missorting in neuronal cultures upon
treatment we calculated the percentage of neurons with missorting in
each eld and normalized the values for the percentage of missorting in
untreated neurons. Signicance between groups was analyzed using the
one-way ANOVA with Newman-Keuls post hoc test; n = 23 replicates
per condition from 3 to 4 independent experiments.
To determine the intensity of Tau and NF200 staining in primary
neuronal cultures, ve individual measures were performed per eld
both for translocated and untranslocated dendrites in a double-blind
quantication of images. Signicance between groups was analyzed at
a distance of 5 μm from cell soma using the one-way ANOVA with
Newman-Keuls post hoc test; n = 23 scannings per condition from 3
independent experiments.
T205-positive signal in granule cell somas and pyramidal dendrites
in CA1 were determined in sections from 1.35 to 2.30 mm posterior to
Bregma. Signicance between groups was analyzed for each area using
the one-way ANOVA with Newman-Keuls post hoc test; n = 35 ani-
mals per genotype. PHF-1-positive mossy bers in the stratum lucidum
was determined in sections from 1.35 to 2.30 mm posterior to Bregma.
Signicance between groups was analyzed using the one-way ANOVA
with Newman-Keuls post hoc test; n = 34 animals per genotype. To
determine the density of PHF-1-positive cells in the hippocampal den-
tate gyrus (from 1.35 mm to 2.30 mm posterior to Bregma), we counted
PHF-1-stained cell somas in the granular layer (GL); data were nor-
malized to the longitude of the GL layer counted in 30-μm-thick sec-
tions containing one hemisphere. Longitudes measured for quantica-
tion were determined using ImageJ on images obtained with NDPview2
from Nanozoomer scans of the preparations. Signicance between
groups was analyzed using the one-way ANOVA with Newman-Keuls
post hoc test; n = 34 animals per genotype.
To determine signicant dierences between groups in the input-
output test, we quantied the slopes of fEPSPs evoked at CA3-
CA1 synapses. fEPSPs were evoked with increasing intensities (from
0.02 to 0.4 mA in steps of 0.02 mA; 10 times for each intensity). Data
evolution and dierences between groups were determined using the
two-way repeated measures ANOVA followed by the Holm-Sidak
method (n 9 animals per genotype). The double-pulse facilitation test
was carried out at the same synapse (n 8 animals per genotype) in
the four groups at six dierent intervals (10 times for each interval).
Again, dierences between groups were determined using the two-way
repeated measures ANOVA followed by the Holm-Sidak method.
Finally, LTP was evoked in the four groups (n = 10 animals per geno-
type) for ve recording sessions following baseline recordings and the
high-frequency stimulation (HFS) train above described. fEPSPs were
evoked at the CA3-CA1 synapse every 20 s and averaged each 5 min
(n = 15 recordings per average) for analysis and representation.
Signicant dierences with baseline recordings and between groups
were determined using the two-way repeated measures ANOVA fol-
lowed by the Holm-Sidak method.
To quantify behavioral performance, distances travelled during the
Open eld test were represented and analyzed separately for the X, Y
and Z axes. Time spent in the central and periphery areas of the cage
were also evaluated. Dierences between groups were determined
using the one-way ANOVA with Newman-Keuls post hoc test. n = 815
animals per genotype. Latencies in the Elevated path were represented
and analyzed using the two-way ANOVA (repeated measures in Test 1
and Test 2 for the same animals) followed by Bonferroni post-test;
n=1014 animals per genotype. Additionally, a Number of fallen and
latency to rst fall in the Rotarod test were represented and analyzed to
determine dierences between groups using the one-way ANOVA with
Newman-Keuls post hoc test; n = 1019 animals per genotype. Passive
Avoidance data were represented as cumulative curves in both times
points of 0 h (habituation) and 24 h and analyzed separately.
Signicance between genotypes was determined by curve comparisons
in pairs using Log-Rank (Mantel Cox) Test for each time point of ana-
lysis; n = 1019 animals per genotype.
3. Results
3.1. Reelin and Tau phosphorylation in mouse models of Tauopathy
To explore the eect of Reelin on Tau phosphorylation in Tauopathy
mouse models, we rst crossbred Reelin-overexpressing mice (TgRln)
(Pujadas et al., 2010;Pujadas et al., 2014) with the Tauopathy mouse
model VLW (overexpressing a 4-repeat isoform of human Tau bearing
three Fronto temporal dementia linked with parkinsonism-17 mutations
(G272 V, P301 L and R406W) (Lim et al., 2001)).
Transgenic mice overexpressing both Reelin and mutated Tau
(VLW), referred to as TgRln/VLW mice, were analyzed for Tau phos-
phorylation levels. In VLW mice, the Tau transgene is directed by in-
sertion of the cDNA into a murine thy1 gene expression cassette and is
thus constitutively expressed in pyramidal neurons distributed across
the cortex and hippocampus, prominently in pyramidal neurons of the
CA1 and in granular cells of the DG, partially overlapping with Reelin
Fig. 1. Reelin reduces Tau phosphorylation in VLW model of Tauopathy.
Hippocampal protein extracts from 6- month-old control, TgRln, VLW and
TgRln/VLW mice were subjected to WB analysis of AD-related phospho-Tau
epitopes T205 (a) and AT8 (b). Reelin overexpression in TgRln/VLW mice
modulates the elevated phosphorylation of the human Tau isoform found in
VLW mice. Changes in phospho-Tau are not due to variations in total Tau
(K9JA) (b). n = 34 animals per genotype. Data are represented as
mean ± SEM; **p< 0.01; the Studentst-test.
D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
5
transgene expression (Suppl. Fig. 1). We performed WB analysis on
hippocampal extracts from TgRln, VLW, and double TgRln/VLW
transgenic mice, as well as on control littermates. Regarding the
transgenic isoform of human Tau (higher molecular weight band), VLW
mice showed a prominent signal of the T205 phosphorylated epitope
(Davila-Bouziguet et al., 2019), while Reelin overexpression led to an
85 % reduction in T205 signal (Fig. 1a). Hyperphosphorylation of an-
other Tau epitope was also tested using the antibody AT8, which re-
cognizes phosphorylation on serines 199202/threonine 205 and has
been associated with early phases of Tauopathy (Wang and Mandelkow,
2016;Bertrand et al., 2010). The AT8 epitope in VLW transgenic ani-
mals showed a non-signicant 60 % decrease when Reelin was co-
overexpressed (Fig. 1b). Finally, total levels of Tau were not sig-
nicantly altered in any genotype by Reelin overexpression, as de-
termined by WB using the phosphorylation independent antibody K9JA
(Fig. 1b).
We also crossbred the TgRln strain with another Tauopathy model,
namely GSK-3βAD mice (Lucas et al., 2001), providing simultaneous
overexpression of Reelin and the Tau kinase GSK-3βin the striatum,
cerebral cortex and hippocampus (Suppl. Fig. 1). In agreement with
previous studies (Wang and Mandelkow, 2016;Lim et al., 2001;
Bertrand et al., 2010;Engel et al., 2006), our data suggest an increase in
Tau phosphorylation in GSK-3βmice that is not observed in TgRln/
GSK-3βmice (Suppl. Fig. 2).
Taken together, our data suggest that Reelin overexpression may
cause a reduction in the extent of Tau phosphorylation in in vivo
Tauopathy models. Given that this was not caused by altered levels of
total Tau, our data suggest a Reelin-dependent reduction of phospho-
Tau levels.
3.2. Reelin reduces Aβ-induced mislocalization of Tau and NF into the
somatodendritic compartment
In addition to altered Tau phosphorylation levels, one of the early
events of Tauopathy is the missorting of endogenous Tau from the axon
to the somatodendritic compartment. This missorting is accompanied
by the redistribution of other axonal cytoskeletal proteins, such as NF,
which also undergoes a loss of polarized distribution. In the context of
AD modeling, mislocalization of axonal proteins can be induced by
amyloid species (Zempel et al., 2013;Zempel et al., 2010). To address
the eect of Reelin on the missorting of axonal cytoskeletal proteins, we
treated mouse primary hippocampal neurons (21 DIV) for 3 h with ei-
ther oligomeric species of Aβ
42
(13μM) in the form of Aβ-derived
diusible ligands (ADDLs) (Lambert et al., 2001) or with vehicle, plus
either puried Reelin or Mock-control. We then examined the sub-
cellular localization of Tau and NF in response to the treatments. The
Fig. 2. Reelin prevents ADDL-induced mislocalization of Tau and neurolaments (NF) into dendrites. Mouse primary hippocampal neurons (21DIV) were
treated with 13μM ADDLs plus 5 μg/mL of Reelin, or equivalent volumes of either vehicle or Mock, or were kept untreated (UT). a) Cells were stained with
phalloidin (red) to visualize dendrites and with anti-Tau (K9JA) (green). UT cells display a diuse signal of Tau in dendrites with no colocalization of intense Tau
signal with phalloidin in dendrites (upper panel). Upon treatment with 1 μM ADDL, an increased proportion of cells show missorted Tau colocalizing with phalloidin
along the dendrites (arrows, middle panel). Reelin signicantly reduces Tau missorting in dendrites (lower panel). b) Cells were stained for the dendrite marker
MAP2 (red) and for NF (NF200) (green) with predominant axonal staining. UT cells display no colocalization of NF200 and MAP2 (upper panel). Upon treatment
with 3 μM ADDL, cultures show an increased proportion of cells with missorted NF colocalizing with MAP2 in dendrites (arrows, middle panel). NF missorting to the
dendrites was prevented in the presence of Reelin (lower panel). c) Quantication of the proportion of neurons showing Tau missorting to dendrites. n = 24
microscopy elds per condition (> 30 neurons/eld) from 4 independent experiments. Proportions of translocated cells were calculated using UT condition in each
experiment for normalization. d) Quantication of the intensity of dendritic Tau staining in both translocated and non-translocated dendrites at the proximal part of
primary dendrites (5 μm from cell soma). n = 5 dendrites per eld, 3 elds per conditions, 3 independent experiments. e) Quantication of the proportion of neurons
displaying neurolament missorting to dendrites. n = 23 microscopy elds per condition (> 30 neurons/eld) from 4 independent experiments. The proportion of
translocated cells was calculated in each condition using UT values for normalization. f) Quantication of the intensity of dendritic NF200 staining in both trans-
located and non-translocated dendrites at the proximal part of primary dendrites (5 μm from cell soma). n = 5 dendrites per eld, 23elds per conditions, 3
independent experiments. Data are presented as mean ± SEM; *p< 0.05; **p< 0.01; ***p< 0.001; one-way ANOVA, Newman-Keuls post hoc test.
D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
6
proportion of Tau and NF missorting was determined by counting the
neurons displaying at least one Tau/NF-translocated dendrite.
Exposure of the cultures to co-treatments of ADDLS (1 μm) and
Reelin (5 μg/mL) led to a signicant decrease in the proportion of
neurons with missorted Tau compared to treatment with ADDLs alone,
thereby indicating that the presence of Reelin caused a reduction in the
missorting phenotype (Fig. 2a,c). A stronger phenotype was observed
with NF missorting upon treatment with ADDLs. Exposure of the cul-
tures to up to 3 μM ADDLs induced a signicant increase in NF mis-
sorting, as compared with vehicle-treated or untreated neurons. Co-
treatment with Reelin (5 μg/mL) completely reversed the proportion of
neurons exhibiting missorted NF (Fig. 2b,e).
To further analyze the eect of Reelin on the distribution of axonal
proteins, the intensity of uorescence signal was quantied in trans-
located dendrites both for NF and Tau proteins. In untreated and mock
neurons, the intensity of the Tau signal in translocated dendrites was
1.5- to 2-fold higher than in neurons with non-translocated Tau
(Fig. 2d,f). The increased intensity of Tau in translocated neurons was
signicantly reduced in neurons treated with Reelin/ADDLs, as com-
pared to those treated with ADDLs alone (Fig. 2f). NF intensity along
translocated dendrites was similar in all the treatment conditions,
thereby indicating that ADDLs and Reelin modulate the number of
neurons displaying translocated NF but do not alter the extent of this
translocation (Fig. 2f).
These results indicate that Reelin rescues in vitro the Aβ-induced
somatodendritic missorting of axonal cytoskeletal proteins such as Tau
and NF. Moreover, Reelin is able to modulate the ne distribution of
Tau protein inside the translocated dendrites, resulting in the reduced
accumulation of this protein in the proximal part of the dendrites.
3.3. Reelin reverts in vivo the somatodendritic localization of
phosphorylated Tau in the hippocampus of the VLW mouse model of
Tauopathy
To analyze the in vivo eect of Reelin on the redistribution of
phospho-Tau, we performed immunohistochemistry with phospho-Tau-
specic antibodies in hippocampal sections from TgRln/VLW mice, as
well as from control, TgRln and VLW littermates.
We rst studied the in vivo distribution of phospho-Tau in the VLW
Tauopathy model and performed immunohistochemistry using the
T205 antibody. Animals from all the genotypes displayed im-
munoreactivity in interneurons distributed throughout all hippocampal
layers. In addition, VLW mice showed an overall increase in phos-
phorylation in the neuropil, and intense somatodendritic staining of
CA1-CA3 pyramidal cells and granule cells in the dentate gyrus, re-
sulting in clear labeling of apical dendrites (Fig. 3a,b,c). This phenotype
was not found in control mice. TgRln/VLW mice showed weaker T205
staining in apical dendrites of CA1 pyramidal cells and in granule cells
(Fig. 3a,b,c). TgRln/VLW mice showed a decrease in the area of stratum
radiatum occupied by T205-stained apical dendrites of CA1 (Fig. 3d,
upper panel). A similar reduction was found in the area occupied by
T205-positive granule cell bodies in TgRln/VLW as compared to VLW
mice (Fig. 3d, lower panel).
Additionally, we also studied the distribution of phospho-Tau in the
GSK-3βAD model using the PHF-1 antibody. GSK-3βanimals showed a
decrease in PHF-1 staining in mossy bers compared to those of Control
and TgRln mice that was not caused by the loss of mossy bers.
Interestingly, we found that TgRln/GSK-3βanimals exhibited a reduc-
tion of the mossy ber phenotype (Suppl. Fig. 3ac). Concomitant with
PHF-1-positive axonal loss, GSK-3βmice showed a signicant increase
in the density of somatodendritic PHF-1-positive cells in the dentate
granular layer that was slightly reduced (not signicant) in TgRln/GSK-
3βmice (Suppl. Fig. 3d,e).
Taken together, our in vitro and in vivo data suggests that Reelin
prevents the somatodendritic missorting of phospho-Tau protein in AD
mouse models.
3.4. Reelin overexpression reverts LTP decits in the VLW Tauopathy model
To unravel the physiological consequences produced by alterations
in Tau, we next determined the modulatory eects of Reelin on the
electrophysiological properties of hippocampal circuits in the VLW
Tauopathy model for the four experimental groups (control, TgRln,
VLW, and TgRln/VLW). For this, we recorded input/output curves,
paired-pulse facilitation, and LTP evoked at the CA3-CA1 synapses in
alert behaving animals (Suppl. Fig. 4)
We analyzed the response of CA1 pyramidal neurons to single pulses
of increasing intensity (0.02-0.4 mA) presented to the ipsilateral CA3
area. As illustrated in Fig. 4a, the four groups of mice presented similar
increases in fEPSP slopes suggesting a normal basal functioning of CA3-
CA1 synapses in all the genotypes analyzed (Madronal et al., 2009)
[F
(57,703)
= 0.518; p = 0.999]. It is generally accepted that changes in
synaptic strength evoked by a pair of pulses are a form of presynaptic
short-term plasticity, mostly related to variations in neurotransmitter
release (Zucker and Regehr, 2002). In particular, paired-pulse stimu-
lation is experimentally used as an indirect measurement of changes in
the probability of neurotransmitter release at presynaptic terminals of
hippocampal synapses (Zucker and Regehr, 2002) even in behaving
mice (Madronal et al., 2009). Paired-pulse facilitation evoked in the
four groups of mice was analyzed presenting a x stimulus intensity
(3040 % of asymptotic values) with increasing inter-pulse intervals
(Fig. 4b). The four groups of mice presented paired pulse facilitations at
short (20 and 40 ms) inter-pulse intervals [F
(5,299)
= 19.635;
p < 0.001]. However, no signicant dierences were observed be-
tween groups [F
(15,230)
= 0.551; p = 0.91].
In a nal experimental step, we carried out an LTP study in the four
groups of mice. As known, CA3-CA1 synapses are involved in the ac-
quisition of dierent types of associative and non-associative learning
tasks and it is usually selected for evoking LTP in behaving mice (Gruart
et al., 2006;Madronal et al., 2007;Madronal et al., 2009). For baseline
values, animals were stimulated in the CA3 area at a rate of 3 times/
min for 15 min (Fig. 4c). Animals were then presented with a HFS
protocol (see Methods). Following the HFS train, the same single sti-
mulus used to generate baseline records was presented at the initial rate
(3/min) for another 60 min. Recording sessions were repeated for four
additional days (30 min each; Fig. 4c). While control and TgRln mice
presented a signicant [F
(114,1368)
= 5.328; p < 0.001] increase in
fEPSP slopes following the HFS session, the VLW mice failed to present
a signicant LTP (p 0.998). This failure was not seen with Reelin
overexpression in TgRln/VLW mice leading to a complex scenario when
VLW Tau mutation is counteracted by Reelin.
In addition, a point to point comparison between fEPSPs evoked in
the four groups of mice after the HFS protocol indicated that the con-
trol, TgRln, and TgRln/VLW groups presented signicantly larger and
longer-lasting (p 0.05) increases in fEPSP values than the VLW group.
Interestingly enough, the TgRln group presented signicantly larger
and longer-lasting increases in fEPSP values than control and TgRln/
VLW groups (Fig. 4c).
In conclusion, the four groups of mice presented similar basal sy-
naptic properties and short-term plasticity. In contrast, VLW mice only
presented a modest and non-signicant LTP and signicantly lower
fEPSP increases in fEPSP values. These LTP decits were not further
observed following overexpression of Reelin in TgRln/VLW mice.
3.5. Reelin overexpression reverts cognitive impairment in the VLW
Tauopathy model
To evaluate the physiological impact of the Reelin-induced mod-
ulations on the phosphorylation and distribution of Tau, animals from
the dierent genotypes (control, TgRln, VLW and TgRln/VLW) were
subjected to behavioral studies. We rst examined motor performance,
observing no relevant dierences between genotypes for the open eld
(Fig. 5a), the rotarod test (Fig. 5b) or the elevated path (Fig. 5c). The
D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
7
cognitive performance of the animals was also evaluated in the fear-
motivated test of passive avoidance. During the acquisition phase of the
test, animals of the dierent genotypes showed identical latencies to
enter to the dark area, with an average time below 20 s (Fig. 5d and
Suppl. Table 1). After 24 h, animals were subjected to the same para-
digm to assess long-term memory. Control mice and TgRln littermates
had an average latency of over 100 s to enter the dark area, thereby
indicating memory retention. In contrast, VLW mice showed lower la-
tencies, thus pointing to impaired memory caused by their Tauopathy
phenotype. Finally, the performance of TgRln/VLW mice, which show
co-expression of Reelin and mutated Tau, was indistinguishable from
that of control mice. This observation thus points to full reversal of the
cognitive alteration in this paradigm upon Reelin overexpression
(Fig. 5d and Suppl. Table 1). These results indicate that VLW mice did
not present evident motor decits; however, they did show signicant
decits in memory retention. In addition, these ndings point to the
physiological relevance of the Reelin-induced reduction in phosphor-
ylation levels and aberrant distribution of Tau in VLW mice. Finally, our
results indicate that Reelin overexpression can counteract Tauopathy-
related histological, electrophysiological and behavioral abnormalities.
4. Discussion
Tau protein is a major player in neurodegeneration and can be
considered a convergent therapeutic target for a diversity of
neurodegenerative diseases. Indeed, alterations in the phosphorylation,
structural conformation and subcellular distribution of Tau are crucial
for the pathogenesis of the most frequent dementias, including AD,
frontotemporal dementias with parkinsonism linked to chromosome 17
(FTDP-17), progressive supranuclear palsy (PSP), corticobasal degen-
eration (CBD), and Picks disease (Wang and Mandelkow, 2016;Buee
and Delacourte, 1999;Gao et al., 2018;Falcon et al., 2018). Regarding
AD, recent evidence has highlighted a major role of Tau in the patho-
genesis of the disease with links to neuroinammation and prion-like
mechanisms (Wang and Mandelkow, 2016;Gao et al., 2018;Goedert
et al., 2017;Hickman et al., 2018;Soto and Pritzkow, 2018). Im-
portantly, Tau is a mediator of Aβtoxicity, as demonstrated in in vitro
and in vivo experiments showing that amyloidosis per se is not patho-
logical in animal models with a Tau knock-out background (Roberson
et al., 2007;Roberson et al., 2011;Chabrier et al., 2012;Chabrier et al.,
2014). In vitro,Aβrequires Tau protein for toxicity and promotes Tau
alterations by altering its phosphorylation state and its ne subcellular
distribution (Li and Gotz, 2017). Moreover, in pathological conditions,
the phosphorylation of Tau at AD-related epitopes (e.g. PHF-1 and AT8)
is believed to start in the axon, resulting in the detachment of Tau from
microtubules and in its redistribution to the somatodendritic compart-
ment (Bertrand et al., 2010;Sohn et al., 2016). This process nally
triggers the destabilization of axons and consequent degeneration
(Kneynsberg et al., 2017). In parallel, the mislocalization of Tau allows
it to mediate Aβ-induced toxicity in the dendritic compartment, thereby
Fig. 3. Reelin reverts the somatodentritic localization of phosphorylated Tau in VLW mice. Immunohistochemistry analysis of phosphorylated Tau (T205) on
hippocampal slices from 6-month-old WT, TgRln, VLW and TgRln/VLW mice. a) In VLW mice, strong somatodendritic immunoreactivity is detected in apical
dendrites from CA1 (black arrowheads) and CA3 (arrows) pyramidal neurons and in somas of the granule cells (white arrowheads). TgRln/VLW mice show decreased
somatodendritic immunoreactivity in all areas, compared to VLW mice. b) Enlarged view of CA1 apical dendrites. c) Enlarged view of granule cells. d) Quantication
of the area of stratum radiatum occupied by T205-stained CA1 apical dendrites (upper panel) and quantication of the area of the granule cell layer occupied by
T205-positive granule cell somas (lower panel). n = 35 animals per genotype. Data are represented as mean ± SEM; *p < 0.05; **p < 0.01; one-way ANOVA,
Newman-Keuls post hoc test. H, hilus; gcl, granular cell layer; ml, molecular layer; so, stratum oriens; sp, stratum piramidale; sr, stratum radiatum; slm, stratum
lacunosum moleculare; sl, stratum lucidum.
D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
8
causing excitotoxic synaptic alterations (Ittner and Gotz, 2011;Ittner
et al., 2010). Indeed, phosphoTau accumulation in the synaptic com-
partment contributes to synaptic dysfunction (Baglietto-Vargas et al.,
2018).
To test the potential of the extracellular protein Reelin as a putative
therapeutic tool for Tauopathy-associated diseases, here we used var-
ious in vivo and in vitro assays of Tau-related pathogenesis (phosphor-
ylation, mislocalization, and cognitive impairment), in a variety of
conditions linked or not to Aβtoxicity. Our results indicates that, in
addition to delaying Aβdeposition in vivo and rescuing synaptic loss
and cognitive impairment in amyloid mouse models (Pujadas et al.,
2014), the overexpression of Reelin counteracts at least two patholo-
gical hallmarks of Tau, namely the somatodendritic missorting of Tau
induced by Aβand the cognitive impairment associated with patholo-
gical Tau mutations in the VLW model. In addition, the present ndings
show a trend for Reelin to reduce Tau phosphorylation. Thus, on the
basis of previous studies (Kocherhans et al., 2010;Doehner et al., 2010;
Knuesel et al., 2009;Krstic et al., 2013) and our own ndings, Reelin
emerges as a possible, convergent therapeutic target in AD-related
diseases by ameliorating both Aβ- and Tau-associated pathologies.
4.1. Reversal of somatodendritic Tau and NF mislocalization by Reelin in
both Aβ-dependent and -independent manners
Our data show that Reelin reverses Aβ-induced NF- and Tau-trans-
location. Indeed, Reelin treatment decreased the proportion of neurons
displaying somatodendritic localization of NF and Tau proteins upon
treatment with ADDLs. However, slight dierences were found in the
degree of rescue for each protein. The signal intensity in proximal
dendrites was modulated by Reelin in the case of Tau, but not in the
case of NF protein, which may be related to the dierent concentrations
used: Tau mislocalization was already induced by incubation with 1 μM
ADDLs, whereas 3 μM treatments were needed to stimulate NF trans-
location. These observations thus suggest that Tau protein shows
greater sensitivity to Aβ-induced mislocalization. It has been shown
that missorted Tau originates from newly synthesized protein in the cell
body (Zempel et al., 2013) and that the aberrant distribution of Tau is
likely to be caused by selective alterations in the axon initial segment, a
key structure in the targeting of neuronal proteins to the axonal and
somatodendritic compartments (Zempel et al., 2017). As Reelin nega-
tively regulates Tau phosphorylation, our results do not allow us to
determine whether the blockade of Tau missorting by Reelin is due to a
direct regulation of the protein targeting mechanisms or, alternatively,
whether it is secondary to the decreased phosphorylation of Tau. Thus,
further studies should address whether decreased Tau phosphorylation
Fig. 4. Reelin overexpression increases LTP in control
animals and reverts LTP decits in the VLW model. a)
Input/output curves of eld excitatory post-synaptic potentials
(fEPSPs) evoked at the CA3-CA1 synapse by single pulses of
increasing intensities (0.02-0.4 in mA) in control (black cir-
cles), TgRln (black squares), VLW (up black triangles), and
TgRln/VLW (down black triangles) (n 9 animals per group).
Data is represented as mean ± SEM. Note the progressive in
fEPSP slopes with the increase in stimulus intensities. No sig-
nicant dierences (p = 0.727; two-way repeated measures
ANOVA) were observed between the four groups. b) Double
pulse facilitation evoked in the four experimental groups (n
8 animals per group). Data shown are mean ± SEM slopes of
the 2nd fEPSP expressed as the percentage of the 1 st for six
(10, 20, 40, 100, 200, 500) inter-pulse intervals. The four
groups presented a facilitation at short (2040 ms) inter-pulse
intervals, with no signicant dierences (p = 0.766; two-way
repeated measures ANOVA) between them. c) Graphs illus-
trating the time course of LTP evoked in the CA3-CA1 synapse
following an HFS session presented to mice included in the
four experimental groups (n = 10 animals per group). After
15 min of baseline recordings, animals were presented with
the high-frequency stimulation (HFS) train described in
Methods and indicated by the dashed line. LTP evolution was
followed for ve days. At the top are illustrated representative
examples of fEPSPs collected at the times indicated in the
bottom graphs collected from a representative animal of each
group. fEPSP slopes are given as a percentage of fEPSP values
collected during baseline recordings (100 %). Three (control,
TgRln, and TgRln/VLW) of the groups presented signicant
(p 0.05) increases in fEPSP values following the HFS, but
not the VLW one (p 0.998). These three groups presented
signicantly (p 0.05) larger fEPSP values than the VLW
group. In addition, the group overexpressing Reelin (TgRln)
also presented signicantly (p 0.05) larger values than
control and TgRln/VLW groups (two-way repeated measures
ANOVA).
D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
9
directly drives or inuences the mislocalization of this protein. Ad-
ditionally, the described interaction of Reelin with ADDLs (Pujadas
et al., 2014) makes it necessary to analyze separately the role of Reelin
in counteracting ADDLs by sequestration and the role of Reelin in di-
rectly modulating Tau localization.
We also explored the mislocalization of Tau in two in vivo models of
Tauopathy in which protein mistargeting is not associated with Aβ
pathology. In the VLW model, the sole expression of human Tau with
epitopes prone to phosphorylation promoted the aberrant distribution
of phospho-Tau to the somatodendritic compartment of the neurons
overexpressing this isoform, i.e., hippocampal pyramidal and granule
cells. The recovered immunohistological distribution of phospho-Tau in
TgRln/VLW mice may be attributable to a reduction in total phospho-
Tau levels as compared to VLW animals. In the GSK-3βmouse model,
the overexpression of this Tau kinase also promoted aberrant phospho-
Tau distribution in the cell bodies of the granular cells of the dentate
gyrus, as described (Lucas et al., 2001). Moreover, here we also show
that the mossy bers show a reduction of phospho-Tau staining that is
rescued by Reelin overexpression. Taken together, the present in vivo
ndings suggest that aberrant phospho-Tau mislocalization is normal-
ized by Reelin.
Our ndings in VLW mice also indicate that the overexpression of
Reelin decreases the levels of Tau hyperphosphorylation caused by the
expression of the human VLW Tau isoform. These ndings were
supported in the mouse model overexpressing the Tau kinase GSK-3β.
These results are consistent with previous studies showing the opposite
eect, i.e. an increase in Tau phosphorylation in heterozygous reeler
mice with reduced Reelin expression or in Reelin receptor knock-outs
(Hiesberger et al., 1999;Kocherhans et al., 2010;Beert et al., 2004).
Taken together, the above results indicate that the Reelin pathway in-
uences the phosphorylation state of Tau protein. Regarding the me-
chanisms of action of Reelin on Tau protein alterations, activation of
the Reelin intracellular cascade, which leads to a decrease in the ac-
tivity of the major kinase for Tau protein, namely GSK-3β(Gonzalez-
Billault et al., 2005;Beert et al., 2002), is likely to contribute to the
observed phosphorylation changes in Tauopathy mouse models over-
expressing Reelin. In addition, the recent nding that the APOE4 pro-
tein, which binds APOER2 receptors, exacerbates Tau pathology (Shi
et al., 2017) suggests that the increase in extracellular Reelin in TgRln
mice may reduce the binding of APOE proteins to the Reelin receptors.
In conclusion, our observations suggest that Reelin could reverse
several hallmarks of Tauopathies, including the following: Tau mis-
localization to the somatodendritic compartment caused by amyloid in
vitro and also in an Aβ-independent manner in mouse models; and al-
tered Tau phosphorylation in vivo. These observations indicate that the
Reelin pathway may interfere with distinct and fundamental molecular
mechanisms associated with Tau pathology.
Fig. 5. Reelin overexpression rescues cognitive decits in
VLW model. a) Open eld. Quantication of the mean dis-
tance travelled by mice (in m) in either X, Y and Z axes inside
the test cage (upper panel). The time (in min) that animals
spent in the central area and in the periphery are also re-
presented (lower panel). Data are represented as
mean ± SEM; no signicant dierences were observed be-
tween groups (one-way ANOVA). n = 919 animals per gen-
otype. b) Accelerating rotarod. Bars show the mean number of
falls (left) and the time (in s; right) that the animals remained
on the rotating bar over the two successive trials. Data are
represented as mean ± SEM; no signicant dierences were
observed between the four experimental groups (one-way
ANOVA). n = 1019 animals per genotype. c) Elevated path
test. The bars indicated the mean latency (in s) to fall of the
four experimental groups during the two trials of the test. Data
are represented as mean ± SEM; no signicant dierences
were observed between groups (two-way ANOVA). n = 1014
animals per genotype. d) Passive avoidance test. Latency to
enter the dark compartment for the control, TgRln, VLW, and
TgRln/VLW groups was measured to a maximum of 180 s.
Data are represented in cumulative frequencies; signicant
dierences between genotypes was determined by curve
comparisons in pairs using Log-Rank (Mantel Cox) Test for
each time point of analysis¸*p < 0.05; **p < 0.01. n = 919
animals per genotype. Although the latency between the four
groups did not dier during the acquisition session, the latency
of the VLW group during the retention session was smaller
than for the other three groups. Indeed, the cumulative fre-
quency presented by the VLW group for the retention test
(24 h) was signicantly smaller than for the other three
groups.
D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
10
4.2. Reelin reverses cognitive and physiological decits in VLW mice
Finally, the present study also explored the physiological relevance
of Reelin on cognitive alterations associated with Tauopathies. Our
results show that, in VLW transgenic mice, Reelin overexpression is
sucient to reverse the cognitive impairment in long-term memory
indicating that the reversal of the Tau-pathogenic molecular and cel-
lular mechanisms described above by Reelin has a direct impact on the
cognitive physiological performance of mouse models of Tauopathy.
Moreover, as already reported (Pujadas et al., 2010), Reelin over-
expression increases dramatically LTP responses in hippocampal cir-
cuits while VLW mice fail to evoke LTP responses at hippocampal CA3-
CA1 synapses. The additive eect of Reelin overexpression in VLW
background led TgRln/VLW mice to behave like controls, a complex
scenario that could either be interpreted as a rescue of the Tauopathy
phenotype in VLW mice or merely as a sum total of antagonistic eects.
5. Conclusions
On the basis of our ndings, we propose that the activation of the
Reelin pathway might provide an ecient therapeutic approach to
ameliorate several pathological mechanisms commonly associated with
Tauopathies, including AD. Thus, on the basis of our previous and
present observations, we conclude that the extracellular protein Reelin
positively targets several amyloid-related pathological processes
(amyloid plaque load, oligomer assembly, synaptic loss, short- and
long-term memory loss and Aβ-induced Tau translocation) (Pujadas
et al., 2014), as well as several Tauopathy-associated processes.
Therefore, the capacity of Reelin as a therapeutic agent targeting both
amyloid- and Tau-associated pathological mechanisms and manifesta-
tions deserves further attention.
Author contributions
E.S and L.P conceived and designed the study, and planned and
supervised the project. DR, AM and LP performed biochemical and
histological experiments and analyzed data. J.A. generated GSK-3βand
VLW transgenic mice. A.G., G.C.-M. and J.M.D.-G. were responsible for
the electrophysiology and behavior experiments. D.R., L.P., E.S.,
J.M.D.-G., A.G. and J.A contributed to writing the manuscript.
Declaration of Competing Interest
Authors declare no conict of interests.
Acknowledgements
This work was supported by grants from La Marató de TV3
Foundation to L.P. and J.M.D.-G. and from MINECO to E.S and L.P.
(SAF2016-76340-R) and to A.G and J.M.D.-G. (BFU2017-82375-R). We
thank Dr. Peter Davies for generously providing PHF-1 antibody; N.
Carulla and A. Vázquez for help in producing ADDLs; M. Sánchez, J.M.
González Martín, A. Lladó and L. Badia for technical assistance; and T.
Yates for editorial help.
Appendix A. The Peer Review Overview and Supplementary data
The Peer Review Overview and Supplementary data associated with
this article can be found in the online version, at doi:https://doi.org/
10.1016/j.pneurobio.2019.101743.
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D. Rossi, et al. Progress in Neurobiology 186 (2020) 101743
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... Putative novel risk SNPs with strong evidence for association were mapped to the DAB1 gene on chromosome 1. Roles for DAB1 and RELN have previously been suggested in AD primarily based on studies in mice ( Hoe et al., 2006 ;Kocherhans et al., 2010 ;Pujadas et al., 2014 ;Rice et al., 2013 ;Rossi et al., 2020 ) and functional genomic analysis in humans ( Gao et al., 2015 ), but genomewide association in humans has been lacking. However, it has been shown that the expression of DAB1 and RELN are altered in AD brains ( Botella-López et al., 2006 ;Chin et al., 2007 ;Muller et al., 2011 ). ...
... In addition, homozygous loss-of-function in Reln and Dab1 have been shown to augment tau-phosphorylation ( Brich et al., 2003 ). Reelin overexpression reduces abnormal somatodendritic localization of phosphor-Tau, A β plaques and synaptic loss in AD model mice ( Pujadas et al., 2014 ;Rossi et al., 2020 ). Thus there are links between the Reelin-DAB1 pathway and the 2 major pathological features of AD. ...
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The APOE-ε4 allele is known to predispose to amyloid deposition and consequently is strongly associated with Alzheimer's disease (AD) risk. There is debate as to whether the APOE gene accounts for all genetic variation of the APOE locus. Another question which remains is whether APOE-ε4 carriers have other genetic factors influencing the progression of amyloid positive individuals to AD. We conducted a genome-wide association study in a sample of 5,390 APOE-ε4 homozygous (ε4ε4) individuals (288 cases and 5,102 controls) aged 65 or over in the UK Biobank. We found no significant associations of SNPs in the APOE locus with AD in the sample of ε4ε4 individuals. However, we identified a novel genome-wide significant locus associated to AD, mapping to DAB1 (rs112437613, OR=2.28, CI=1.73-3.01, p=5.4 × 10⁻⁹). This identification of DAB1 led us to investigate other components of the DAB1-RELN pathway for association. Analysis of the DAB1-RELN pathway indicated that the pathway itself was associated with AD, therefore suggesting an epistatic interaction between the APOE locus and the DAB1-RELN pathway.
... The effects of Reelin overexpression on the pathology of tauopathy were investigated using AD-related mice expressing human mutant Tau (G272V, P301L and R406W), which are called VLW mice [103]. Increases in Tau phosphorylation levels in the hippocampus of VLW mice were reduced by the overexpression of Reelin. ...
... These findings suggest that enhancements in Reelin signaling protect against the symptoms of Tau pathology. Therefore, Reelin may be a therapeutic target in AD [103]. ...
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Reelin is an extracellular matrix protein that is mainly produced in Cajal-Retzius cells and controls neuronal migration, which is important for the proper formation of cortical layers in the developmental stage of the brain. In the adult brain, Reelin plays a crucial role in the regulation of N-methyl-D-aspartate receptor-dependent synaptic function, and its expression decreases postnatally. Clinical studies showed reductions in Reelin protein and mRNA expression levels in patients with psychiatric disorders; however, the causal relationship remains unclear. Reelin-deficient mice exhibit an abnormal neuronal morphology and behavior, while Reelin supplementation ameliorates learning deficits, synaptic dysfunctions, and spine loss in animal models with Reelin deficiency. These findings suggest that the neuronal deficits and brain dysfunctions associated with the down-regulated expression of Reelin are attenuated by enhancements in its expression and functions in the brain. In this review, we summarize findings on the role of Reelin in neuropsychiatric disorders and discuss potential therapeutic approaches for neuropsychiatric disorders associated with Reelin dysfunctions.
... A few negative values occurred, and these were set to zero. Given the substantial biological variability in the physiological levels of reelin and iAβ (38,39), data from each single animal and both conditions were normalized, by linearly mapping the fluorescence level in the [0,1] interval (75,76). The normalized data from all animals were then pooled together, while keeping track of the preprint (which was not certified by peer review) is the author/funder. ...
... A converse situation is apparent when mating transgenic mice overexpressing reelin, with transgenic mice expressing mutated human tau that normally develop ample p-tau by 6 months. The resulting offspring exhibit a dramatic reduction of p-tau (76). Furthermore, the application of reelin-Aβ aggregates onto mouse primary neurons result in a two-fold increase in levels of p-tau in the cell-extracts relative to that seen following the addition of pure reelin (59). ...
Preprint
Projection neurons in the anterolateral part of entorhinal cortex layer II (alEC LII) are the predominant cortical site for hyperphosphorylation of tau (p-tau) and formation of neurofibrillary tangles (NFTs) in brains of subjects with early-stage Alzheimer’s Disease (AD). A majority of alEC LII-neurons are unique among cortical excitatory neurons by expressing the protein reelin (Re+). In AD patients, and a rat model for AD overexpression mutated human APP, these Re+ excitatory projection-neurons are prone to accumulate intracellular amyloid-β (iAβ). Biochemical pathways that involve reelin-signaling regulate levels of p-tau, and iAβ has been shown to impair such reelin-signaling. We therefore used the rat model and set out to assess whether accumulation of iAβ in Re+ alEC LII projection neurons relates to the fact that these neurons express reelin. Here we show that in Re+ alEC LII-neurons, reelin and iAβ42 engage in a direct protein-protein interaction, and that microRNA-mediated lowering of reelin-levels in these neurons leads to a concomitant reduction of non-fibrillar iAβ ranging across three levels of aggregation. Our experiments are carried out several months before plaque pathology emerges in the rat model, and the reduction of iAβ occurs without any substantial associated changes in human APP-levels. We propose a model positioning reelin in a sequence of changes in functional pathways in Re+ alEC LII-neurons, explaining the region and neuron-specific initiation of AD pathology. Significance Anterolateral entorhinal cortex layer II (EC LII) neurons are the predominant cortical site for hyperphosphorylation of tau (p-tau) and formation of neurofibrillary tangles (NFTs) in brains of subjects with early-stage Alzheimer’s disease (AD). The same neurons are prone to very early accumulation of non-fibrillary forms of amyloid-β in the context of AD, and are unique among cortical excitatory neurons by expressing the protein reelin. We show that in such alEC LII-neurons, reelin and iAβ42 engage in a direct protein-protein interaction, and that selectively lowering levels of reelin leads to a concomitant reduction of non-fibrillar Aβ. We propose a model positioning reelin in a sequence of changes in functional pathways in reelin-expressing EC LII neurons, explaining the region and neuron specific initiation of AD.
... Modification of PNNs in Alzheimer's disease could also come about through the action of activated microglia or secretion of metalloproteinases, both of which can occur in this condition [191,194]. Reelin is an ECM-associated protein with effects on plasticity, and overexpression of this molecule restores memory in a tauopathy model [195]. Although much is yet to be understood about the role of PNNs in Alzheimer's disease progression and cognitive decline, these investigations point important and mostly uncovered territory to understand this disease and identify muchneeded new drug targets. ...
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All components of the CNS are surrounded by a diffuse extracellular matrix (ECM) containing chondroitin sulphate proteoglycans (CSPGs), heparan sulphate proteoglycans (HSPGs), hyaluronan, various glycoproteins including tenascins and thrombospondin, and many other molecules that are secreted into the ECM and bind to ECM components. In addition, some neurons, particularly inhibitory GABAergic parvalbumin-positive (PV) interneurons, are surrounded by a more condensed cartilage-like ECM called perineuronal nets (PNNs). PNNs surround the soma and proximal dendrites as net-like structures that surround the synapses. Attention has focused on the role of PNNs in the control of plasticity, but it is now clear that PNNs also play an important part in the modulation of memory. In this review we summarize the role of the ECM, particularly the PNNs, in the control of various types of memory and their participation in memory pathology. PNNs are now being considered as a target for the treatment of impaired memory. There are many potential treatment targets in PNNs, mainly through modulation of the sulphation, binding, and production of the various CSPGs that they contain or through digestion of their sulphated glycosaminoglycans.
... In experimental models, Reelin-ApoE receptor-Dab1 signaling stabilizes microtubules via a kinase cascade involving Tyr607-pPI3K-induced activation of Akt-mediated Ser9-phosphorylation and inhibition of GSK3β, which suppresses tau phosphorylation (Fig. 1B). Compromised Reelin signaling through ApoE receptors promotes GSK3β-mediated tau hyperphosphorylation and somatodendritic localization [40,42,139,140], which can be reversed by Reelin overexpression [141], implying a direct link between deficits in Reelin-ApoE receptor signaling and this defining AD pathology. This interpretation, when considered together with our observations that Reelin, ApoER2, Dab1, and Thr508-pLIMK1 accumulate in close proximity to Tyr607-pPI3K and Ser202/Thr205-Tau in a subset of neuritic plaques (Figs. ...
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Background: Sporadic Alzheimer's disease (sAD) lacks a unifying hypothesis that can account for the lipid peroxidation observed early in the disease, enrichment of ApoE in the core of neuritic plaques, hallmark plaques and tangles, and selective vulnerability of entorhinal-hippocampal structures. Objective: We hypothesized that 1) high expression of ApoER2 (receptor for ApoE and Reelin) helps explain this anatomical vulnerability; 2) lipid peroxidation of ApoE and ApoER2 contributes to sAD pathogenesis, by disrupting neuronal ApoE delivery and Reelin-ApoER2-Dab1 signaling cascades. Methods: In vitro biochemical experiments; Single-marker and multiplex fluorescence-immunohistochemistry (IHC) in postmortem specimens from 26 individuals who died cognitively normal, with mild cognitive impairment or with sAD. Results: ApoE and ApoER2 peptides and proteins were susceptible to attack by reactive lipid aldehydes, generating lipid-protein adducts and crosslinked ApoE-ApoER2 complexes. Using in situ hybridization alongside IHC, we observed that: 1) ApoER2 is strongly expressed in terminal zones of the entorhinal-hippocampal 'perforant path' projections that underlie memory; 2) ApoE, lipid aldehyde-modified ApoE, Reelin, ApoER2, and the downstream Reelin-ApoER2 cascade components Dab1 and Thr19-phosphorylated PSD95 accumulated in the vicinity of neuritic plaques in perforant path terminal zones in sAD cases; 3) several ApoE/Reelin-ApoER2-Dab1 pathway markers were higher in sAD cases and positively correlated with histological progression and cognitive deficits. Conclusion: Results demonstrate derangements in multiple ApoE/Reelin-ApoER2-Dab1 axis components in perforant path terminal zones in sAD and provide proof-of-concept that ApoE and ApoER2 are vulnerable to aldehyde-induced adduction and crosslinking. Findings provide the foundation for a unifying hypothesis implicating lipid peroxidation of ApoE and ApoE receptors in sAD.
... Among different ECM components, Reelin can regulate the migration of neurons during the developmental period of the brain. This protein is touted as a key player in the formation of the cerebral cortex and lamination of the cerebellum (Rossi et al., 2020), and maintenance of adult synaptogenesis (Pujadas et al., 2010). Likewise, it was suggested that Reelin can reduce pathologies related to cerebral ischemia-reperfusion injury. ...
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Abstract Ischemic stroke is characterized by extensive neuronal loss, glial scar formation, neural tissue degeneration that leading to profound changes in the extracellular matrix, neuronal circuitry, and long‐lasting functional disabilities. Although transplanted neural stem cells (NSCs) can recover some of the functional deficit after stroke, retrieval is not complete and repair of lost tissue is negligible. Therefore, the current challenge is to use the combination of NSCs with suitably enriched biomaterials to retain these cells within the infarct cavity and accelerate the formation of a de novo tissue. This study aimed to test the regenerative potential of polylactic‐co‐glycolic acid‐polyethylene glycol (PLGA‐PEG) micelle biomaterial enriched with Reelin and embryonic NSCs on photothrombotic stroke model of mice to gain appropriate methods in tissue engineering. For this purpose, two sets of experiments, either in vitro or in vivo models, were performed. In vitro analyses exhibited PLGA‐PEG plus Reelin‐induced proliferation rate (Ki‐67+ NSCs) and neurite outgrowth (axonization and dendritization) compared to PLGA‐PEG + NSCs and Reelin + NSCs groups (p
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Aluminum (Al) is a common neurotoxic element that can exacerbate intracellular β-amyloid (Aβ) deposition. Reelin is a highly conserved extracellular glycoprotein that is involved in intracellular Aβ deposition. However, the action of Reelin on aluminum-induced Aβ deposition is not fully understood. Here, we investigated the effects of the Reelin-Dab1 signaling pathway on Aβ deposition in aluminum maltol (Al(mal)3) exposure in rat pheochromocytoma-derived cells (PC12). Our results showed that Al(mal)3 exposure decreased activity of PC12, increased expression of Aβ42, and decreased expression of Aβ40. Moreover, Al(mal)3 exposure in PC12 induced Reelin-Dab1 signaling pathway-associated proteins changed, decreased expression of Reelin and Dab1, and increased expression of pdab1. Moreover, the expression of Reelin, Dab1, and Aβ40 was found to be elevated in PC12 exposed to Al(mal)3 and corticosterone compared to those exposed to Al(mal)3. Also, the expression of Reelin, Dab1, and Aβ40 was found to be depressed in PC12 exposed to Al(mal)3 and streptozotocin compared with cells exposed to Al(mal)3 alone. These results suggested that Al(mal)3 inhibits the expression of the Reelin-Dab1 signaling pathway, promoting Aβ deposition. Thus, our findings provided important evidence to better understand how the Reelin-Dab1 signaling pathway may be a potential mechanism of Aβ deposition induced by aluminum.
Chapter
Biomarkers of PTSD are greatly needed because of the challenges to diagnose and treat due to stigma, biases in self-reporting, and limitations of identifying those at risk and the scarcity of tools to predict treatment outcome. PTSD is influenced by both genetic and environmental factors as well as its interplay. Because of the dynamic and context-dependent nature of epigenetic modifications, these offer great promise as biomarkers of PTSD for the prevention, prognosis, and prediction of treatment outcome. In this chapter, genomic and epigenomic studies of PTSD are discussed. Epigenomic studies of PTSD conducted to date include DNA methylation (including DNA methylation age), histone modifications, and noncoding RNAs. Further, we discuss recent work using integrative multiomics approach as well as related gaps, challenges, and future directions aimed at identifying biomarkers of PTSD.
Chapter
Posttraumatic stress disorder (PTSD) is an acquired, debilitating, psychiatric disorder characterized by functionally deficient physiological and psychological symptoms in the aftermath of a traumatic event. This chapter emphasizes the critical role of pharmacoepigenetics in addressing the interindividual variability seen in patients treated with current medications for trauma- and stress-related neuropsychiatric disorders such as PTSD, and highlights the scarcity of effective drugs for treating the symptoms of patients with these disorders. Chronic PTSD treatment is complicated by comorbid symptomatology, and attempts to suppress traumatic memories, for example, remain difficult. As the epigenome can either modulate drug response or be pharmacologically regulated, we underline the importance of epigenetic-based interventions for alleviating PTSD pathogenesis. We hypothesize that selective epigenetic machinery targeting with epidrugs and/or epinutraceuticals could be a viable strategy to mitigate trauma- and stress-related neuropsychiatric disorders. Furthermore, compelling evidence has linked immune system dysregulation to abnormal neuronal signaling and psychiatric disorders. However, much effort is required to transform and advance our understanding of PTSD and comorbid disorders so that new and innovative interventions for those suffering from this critical neuropsychiatric illness can be developed.
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The medial temporal lobe memory system has long been identified as the brain region showing the first histopathological changes in early Alzheimer’s disease (AD), and the functional decline observed in patients also points to a loss of function in this brain area. Nonetheless, the exact identity of the neurons and networks that undergo deterioration has not been determined so far. A recent study has identified the entorhinal and hippocampal neural circuits responsible for encoding new episodic memories. Using this novel model we describe the elements of the episodic memory network that are especially vulnerable in early AD. We provide a hypothesis of how reduced reelin signaling within such a network can promote AD-related changes. Establishing novel associations and creating a temporal structure for new episodic memories are both affected in AD. Here, we furnish a reasonable explanation for both of these previous observations.
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The neuroimmune system is involved in development, normal functioning, aging, and injury of the central nervous system. Microglia, first described a century ago, are the main neuroimmune cells and have three essential functions: a sentinel function involved in constant sensing of changes in their environment, a housekeeping function that promotes neuronal well-being and normal operation, and a defense function necessary for responding to such changes and providing neuroprotection. Microglia use a defined armamentarium of genes to perform these tasks. In response to specific stimuli, or with neuroinflammation, microglia also have the capacity to damage and kill neurons. Injury to neurons in Alzheimer's, Parkinson's, Huntington's, and prion diseases, as well as in amyotrophic lateral sclerosis, frontotemporal dementia, and chronic traumatic encephalopathy, results from disruption of the sentinel or housekeeping functions and dysregulation of the defense function and neuroinflammation. Pathways associated with such injury include several sensing and housekeeping pathways, such as the Trem2, Cx3cr1 and progranulin pathways, which act as immune checkpoints to keep the microglial inflammatory response under control, and the scavenger receptor pathways, which promote clearance of injurious stimuli. Peripheral interference from systemic inflammation or the gut microbiome can also alter progression of such injury. Initiation or exacerbation of neurodegeneration results from an imbalance between these microglial functions; correcting such imbalance may be a potential mode for therapy.
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The ordered assembly of tau protein into abnormal filamentous inclusions underlies many human neurodegenerative diseases1. Tau assemblies seem to spread through specific neural networks in each disease2, with short filaments having the greatest seeding activity3. The abundance of tau inclusions strongly correlates with disease symptoms4. Six tau isoforms are expressed in the normal adult human brain-three isoforms with four microtubule-binding repeats each (4R tau) and three isoforms that lack the second repeat (3R tau)1. In various diseases, tau filaments can be composed of either 3R or 4R tau, or of both. Tau filaments have distinct cellular and neuroanatomical distributions5, with morphological and biochemical differences suggesting that they may be able to adopt disease-specific molecular conformations6,7. Such conformers may give rise to different neuropathological phenotypes8,9, reminiscent of prion strains10. However, the underlying structures are not known. Using electron cryo-microscopy, we recently reported the structures of tau filaments from patients with Alzheimer's disease, which contain both 3R and 4R tau11. Here we determine the structures of tau filaments from patients with Pick's disease, a neurodegenerative disorder characterized by frontotemporal dementia. The filaments consist of residues Lys254-Phe378 of 3R tau, which are folded differently from the tau filaments in Alzheimer's disease, establishing the existence of conformers of assembled tau. The observed tau fold in the filaments of patients with Pick's disease explains the selective incorporation of 3R tau in Pick bodies, and the differences in phosphorylation relative to the tau filaments of Alzheimer's disease. Our findings show how tau can adopt distinct folds in the human brain in different diseases, an essential step for understanding the formation and propagation of molecular conformers.
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Alzheimer's disease (AD) is a devastating neurodegenerative disorder that impairs memory and causes cognitive and psychiatric deficits. New evidences indicate that AD is conceptualized as a disease of synaptic failure, although the molecular and cellular mechanisms underlying these defects remain to be elucidated. Determining the timing and nature of the early synaptic deficits is critical for understanding the progression of the disease and for identifying effective targets for therapeutic intervention. Using single‐synapse functional and morphological analyses, we find that AMPA signaling, which mediates fast glutamatergic synaptic transmission in the central nervous system (CNS), is compromised early in the disease course in an AD mouse model. The decline in AMPA signaling is associated with changes in actin cytoskeleton integrity, which alters the number and the structure of dendritic spines. AMPA dysfunction and spine alteration correlate with the presence of soluble but not insoluble Aβ and tau species. In particular, we demonstrate that these synaptic impairments can be mitigated by Aβ immunotherapy. Together, our data suggest that alterations in AMPA signaling and cytoskeletal processes occur early in AD. Most important, these deficits are prevented by Aβ immunotherapy, suggesting that existing therapies, if administered earlier, could confer functional benefits.
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The ordered assembly of tau protein into abnormal filamentous inclusions underlies many human neurodegenerative diseases. Tau assemblies appear to spread through specific neural networks in each disease, with short filaments having the greatest seeding activity. The abundance of tau inclusions strongly correlates with disease symptoms. Six tau isoforms are expressed in normal adult human brain - three isoforms with four microtubule-binding repeats each (4R tau) and three isoforms lacking the second repeat (3R tau). In various diseases, tau filaments can be composed of either 3R tau or 4R tau, or of both 3R and 4R tau. They have distinct cellular and neuroanatomical distributions, with morphological and biochemical differences suggesting that they may be able to adopt disease-specific molecular conformations. Such conformers may give rise to different neuropathological phenotypes, reminiscent of prion strains. However, the underlying structures are not known. Using electron cryo-microscopy (cryo-EM), we recently reported the structures of tau filaments from Alzheimer's disease, which contain both 3R and 4R tau. Here we have determined the structures of tau filaments from Pick's disease, a neurodegenerative disorder characterised by frontotemporal dementia. They consist of residues K254-F378 of 3R tau, which are folded differently when compared to tau in Alzheimer's disease filaments, establishing the existence of conformers of assembled tau. The Pick fold explains the selective incorporation of 3R tau in Pick bodies and the differences in phosphorylation relative to the tau filaments of Alzheimer's disease. Our findings show how tau can adopt distinct folds in human brain in different diseases, an essential step for understanding the formation and propagation of molecular conformers.
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Tauopathies are a diverse group of diseases featuring progressive dying-back neurodegeneration of specific neuronal populations in association with accumulation of abnormal forms of the microtubule-associated protein tau. It is well-established that the clinical symptoms characteristic of tauopathies correlate with deficits in synaptic function and neuritic connectivity early in the course of disease, but mechanisms underlying these critical pathogenic events are not fully understood. Biochemical in vitro evidence fueled the widespread notion that microtubule stabilization represents tau's primary biological role and that the marked atrophy of neurites observed in tauopathies results from loss of microtubule stability. However, this notion contrasts with the mild phenotype associated with tau deletion. Instead, an analysis of cellular hallmarks common to different tauopathies, including aberrant patterns of protein phosphorylation and early degeneration of axons, suggests that alterations in kinase-based signaling pathways and deficits in axonal transport (AT) associated with such alterations contribute to the loss of neuronal connectivity triggered by pathogenic forms of tau. Here, we review a body of literature providing evidence that axonal pathology represents an early and common pathogenic event among human tauopathies. Observations of axonal degeneration in animal models of specific tauopathies are discussed and similarities to human disease highlighted. Finally, we discuss potential mechanistic pathways other than microtubule destabilization by which disease-related forms of tau may promote axonopathy.
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APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-β pathology relative to other ApoE isoforms¹. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-α (TNF-α) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-α. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an ε4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-β pathology with symptomatic Alzheimer disease who usually have tau pathology, ε4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-β pathology. ApoE4 exerts a ‘toxic’ gain of function whereas the absence of ApoE is protective.
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Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β peptide (Aβ) and hyperphosphorylated Tau protein (P-Tau). Our recent data showed a differential accumulation of Tau protein phosphorylated at residue Thr231 (pThr231) in distinct hippocampal neurons in VLW mice—a model that overexpresses mutated human Tau. Here we demonstrate that, in VLW mice, the accumulation of human P-Tau in pyramidal cells induces the phosphorylation of murine Tau at residue Thr231 in hippocampal interneurons. In addition, we show that pSer262 and pThr205 Tau are present specifically in the soma of some hippocampal interneurons in control mice. Analysis of J20 mice—a model that accumulates Aβ—and of VLW animals showed that the density of hippocampal interneurons accumulating pThr205 Tau is lower in VLW mice than in controls. In contrast, the density of interneurons accumulating pThr205 Tau in J20 mice was increased compared to controls in hippocampal regions with a higher Aβ plaque load, thereby suggesting that pThr205 Tau is induced by Aβ. No significant differences were found between the density of hippocampal interneurons positive for pSer262 Tau in VLW or J20 mice compared to control animals. We also show that pSer262 and pThr205 Tau are present in the soma of some hippocampal interneurons containing Parvalbumin, Calbindin or Calretinin in control, VLW, and J20 mice. Moreover, our results reveal that some interneurons in human hippocampi of cases of AD and control cases accumulate pSer262 and pThr205 Tau. Taken together, these data point to a specific role of pSer262 and pThr205 Tau in the soma of hippocampal interneurons in control and pathological conditions.
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Technologies for imaging the pathophysiology of Alzheimer disease (AD) now permit studies of the relationships between the two major proteins deposited in this disease - amyloid-β (Aβ) and tau - and their effects on measures of neurodegeneration and cognition in humans. Deposition of Aβ in the medial parietal cortex appears to be the first stage in the development of AD, although tau aggregates in the medial temporal lobe (MTL) precede Aβ deposition in cognitively healthy older people. Whether aggregation of tau in the MTL is the first stage in AD or a fairly benign phenomenon that may be transformed and spread in the presence of Aβ is a major unresolved question. Despite a strong link between Aβ and tau, the relationship between Aβ and neurodegeneration is weak; rather, it is tau that is associated with brain atrophy and hypometabolism, which, in turn, are related to cognition. Although there is support for an interaction between Aβ and tau resulting in neurodegeneration that leads to dementia, the unknown nature of this interaction, the strikingly different patterns of brain Aβ and tau deposition and the appearance of neurodegeneration in the absence of Aβ and tau are challenges to this model that ultimately must be explained.
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A hallmark event in neurodegenerative diseases (NDs) is the misfolding, aggregation, and accumulation of proteins, leading to cellular dysfunction, loss of synaptic connections, and brain damage. Despite the involvement of distinct proteins in different NDs, the process of protein misfolding and aggregation is remarkably similar. A recent breakthrough in the field was the discovery that misfolded protein aggregates can self-propagate through seeding and spread the pathological abnormalities between cells and tissues in a manner akin to the behavior of infectious prions in prion diseases. This discovery has vast implications for understanding the mechanisms involved in the initiation and progression of NDs, as well as for the design of novel strategies for treatment and diagnosis. In this Review, we provide a critical discussion of the role of protein misfolding and aggregation in NDs. Commonalities and differences between distinct protein aggregates will be highlighted, in addition to evidence supporting the hypothesis that misfolded aggregates can be transmissible by the prion principle. We will also describe the molecular basis and implications for prion-like conformational strains, cross-interaction between different misfolded proteins in the brain, and how these concepts can be applied to the development of novel strategies for therapy and diagnosis.
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Tau, a microtubule-associated protein, is the main component of the intracellular filamentous inclusions that are involved in neurodegenerative diseases known as tauopathies, including Alzheimer disease (AD), frontotemporal dementia with parkinsonism-17 (FTDP-17), Pick disease (PiD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Hyperphosphorylated, aggregated tau proteins form the core of neurofibrillary tangles (NFTs), which are shown to be one of the pathological hallmarks of AD. The discovery of mutations in the microtubule-associated protein tau (MAPT) gene in patients with FTDP-17 also contributes to a better understanding of the dysfunctional tau as a cause of diseases. Although recent substantial progress has been made in the tau pathology of tauopathies, the mechanisms underlying tau-induced neurodegeneration remain unclear. Here, we present an overview of the biochemical properties of tau protein and the pathogenesis underlying tau-induced neurodegenerative diseases. Meanwhile, we will discuss the tau-related biomarkers and ongoing tau-targeted strategies for therapeutic modulation.