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\begin{document}

\title{Disease-modifying treatment with I2 imidazoline receptor ligand LSL60101
in an Alzheimer's disease mouse model : A Comparative study with
donepezil}



\author[1]{Fotini Vasilopoulou}%
\author[1]{Sergio Rodríguez-Arevalo}%
\author[1]{Andrea Bagán}%
\author[1]{Carmen Escolano}%
\author[1]{Christian Griñán-Ferré}%
\author[1]{Mercè Pallàs}%
\affil[1]{Universitat de Barcelona Facultat de Farmacia}%


\vspace{-1em}



  
  \date{March 13, 2025}


\begingroup
\let\center\flushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
Background and Purpose: The development of effective therapeutic
strategies against Alzheimer's disease (AD) remains a challenge. I2
Imidazoline receptors (I2-IR) ligands have a neuroprotective role in AD.
While co-treatment of acetylcholinesterase inhibitors with
neuroprotective agents have shown better effects on the prevention of
dementia. Here, we assessed the potential therapeutic effect of the
I2-IR ligand LSL60101, donepezil and their combination in 5XFAD mice.
Experimental Approach: 5XFAD female mice were treated with low doses of
LSL60101 (1mg/kg/day), donepezil (1mg/kg/day), and donepezil plus
LSL60101 (1+1mg/kg/day), during 4 weeks per os. Novel object
recognition, Morris water maze, open field, elevated plus maze and
three-chamber tests were employed to evaluate the cognitive and
behavioural status of the mice after treatment. The effects of the
treatments on AD-like pathology were assessed with immunohistochemistry,
Western blot and qPCR. Key results: Chronic low-dose treatment with
LSL60101 and donepezil reversed cognitive deficits and impaired social
behaviour. LSL60101 treatment did not affect anxiety-like behaviour in
contrast to donepezil. In the 5XFAD brains, LSL60101 and
donepezil/LSL60101 treatments decreased A\selectlanguage{greek}β-\selectlanguage{english}pathology and Tau
hyperphosphorylation, and these alterations were accompanied by
decreased microglia marker Iba-1 levels and increased Trem2 gene
expression. LSL60601 and donepezil decreased glial fibrillary acidic
protein (GFAP) astrocytic marker reactivity. However, only LSL60601
treatment significantly increased the levels of the synaptic markers
post-density 95 (PSD95) and synaptophysin (SYN). Conclusion and
implications: Our results suggest that chronic low dose treatment with
selective I2-IR ligands can be an effective treatment for AD and provide
insights into combination treatments of symptomatic and
disease-modifying drugs%
\end{abstract}%



\sloppy


\textbf{Disease-modifying treatment with I2 imidazoline receptor ligand
LSL60101 in an Alzheimer's disease mouse model: A Comparative study with
donepezil.}

\emph{Running Title:} I2 Imidazoline receptor modulation as a potential
therapeutic strategy against Alzheimer's Disease.

Foteini Vasilopoulou\textsuperscript{1}, Sergio
Rodr\selectlanguage{ngerman}íguez-Arévalo\textsuperscript{2}, Andrea Bagán\textsuperscript{2},
Carmen Escolano\textsuperscript{2}, Christian
Griñán-Ferré\textsuperscript{1*} and Mercè Pallàs\textsuperscript{1}*

\textsuperscript{1}Pharmacology Section. Department of Pharmacology,
Toxicology and Medicinal Chemistry, Faculty of Pharmacy and Food
Sciences, and Institut de Neurociències, University of Barcelona, Av.
Joan XXIII, 27-31, E-08028 Barcelona, Spain.

\textsuperscript{2}Laboratory of Medicinal Chemistry (Associated Unit to
CSIC), Department of Pharmacology, Toxicology and Medicinal Chemistry,
Faculty of Pharmacy and Food Sciences, and Institute of Biomedicine
(IBUB), University of Barcelona, Av. Joan XXIII, 27-31, E-08028
Barcelona, Spain.

\textbf{*Corresponding authors}

\textbf{WORD COUNT:} Main text 6257 words (Introduction, methods,
results and discussion) and 3513 words (excluding methods, references
and figure legends)

\textbf{ACKNOWLEDGEMENTS}

This study was supported by Ministerio de Economía y Competitividad of
Spain and FEDER

(PID2019-107991RB-I00, PID2019-106285RB-I00) and 2017SGR106 (AGAUR,
Catalonia). The project leading to these results has received funding
from ``la Caixa'' Foundation (ID100010434), under agreement CI18-00002.
F.V. thanks the UB for APIF grant (UB2016); S. R.-A. to Generalitat de
Catalunya, (2018FI-B-00227) and A. B. for APIF Grant Institute of
Biomedicine (UB2018).

\textbf{AUTHOR CONTRIBUTIONS}

FV, CG-F and MP conceived the study, designed all the experiments, and
interpreted the data; FV, CG-F performed the experiments and data
analysis; all authors revised the manuscript draft. S R-A, AB and CE
synthesized compounds tested; FV, CG-F and MP wrote, revised, and
finalised the manuscript.

\subsection*{CONFLICT OF INTEREST}

{\label{conflict-of-interest}}

The authors declare no conflicts of interest.

\textbf{DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR}

This Declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical research
as stated in the~\emph{BJP} ~guidelines for~\emph{Design \&
Analysis},~\emph{Immunoblotting and Immunochemistry}~and~\emph{Animal
Experimentation}~and as recommended by funding agencies, publishers and
other organizations engaged with supporting research.

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding author upon reasonable request. Some data may not be
available because of privacy or ethical restrictions.

\textbf{ABSTRACT}

\emph{Background and Purpose\textbf{:}} The development of effective
therapeutic strategies against Alzheimer's disease (AD) remains a
challenge. I2 Imidazoline receptors (I2-IR) ligands have a
neuroprotective role in AD. While co-treatment of acetylcholinesterase
inhibitors with neuroprotective agents have shown better effects on the
prevention of dementia. Here, we assessed the potential therapeutic
effect of the I2-IR ligand LSL60101, donepezil and their combination in
5XFAD mice.

\emph{Experimental Approach} : 5XFAD female mice were treated with low
doses of LSL60101 (1 mg\textsuperscript{-1}
kg\textsuperscript{-1}day\textsuperscript{-1}), donepezil (1
mg\textsuperscript{-1}kg\textsuperscript{-1} day\textsuperscript{-1}),
and donepezil plus LSL60101 (1+1 mg\textsuperscript{-1}
kg\textsuperscript{-1}day\textsuperscript{-1}), during 4 weeks \emph{per
os} . Novel object recognition, Morris water maze, open field, elevated
plus maze and three-chamber tests were employed to evaluate the
cognitive and behavioural status of the mice after treatment. The
effects of the treatments on AD-like pathology were assessed with
immunohistochemistry, Western blot and qPCR.

\emph{Key results:} Chronic low-dose treatment with LSL60101 and
donepezil reversed cognitive deficits and impaired social behaviour.
LSL60101 treatment did not affect anxiety-like behaviour in contrast to
donepezil. In the 5XFAD brains, LSL60101 and donepezil/LSL60101
treatments decreased A\selectlanguage{greek}β-\selectlanguage{english}pathology and Tau hyperphosphorylation, and
these alterations were accompanied by decreased microglia marker Iba-1
levels and increased \emph{Trem2} gene expression. LSL60601 and
donepezil decreased glial fibrillary acidic protein (GFAP) astrocytic
marker reactivity. However, only LSL60601 treatment significantly
increased the levels of the synaptic markers post-density 95 (PSD95) and
synaptophysin (SYN).

\emph{Conclusion and implications:} Our results suggest that chronic low
dose treatment with selective I2-IR ligands can be an effective
treatment for AD and provide insights into combination treatments of
symptomatic and disease-modifying drugs.

\textbf{KEYWORDS:} I2 Imidazoline receptors, \selectlanguage{greek}β-\selectlanguage{english}amyloid,
neuroinflammation, synaptic plasticity, donepezil, Alzheimer's disease

\textbf{BULLET POINT SUMMARY}

\emph{What is already known}

I2-IR modulation by selective I2-IR ligands delivers neuroprotection in
the central nervous system.

AchEI did not modify disease progressions, thereby a new drug for
halting AD progression is needed.

\emph{What this study adds}

LSL60101, an I2-IR ligand, treatment rescued 5XFAD mice from cognitive
impairments and modified disease progression.

LSL60101 treatment provides greater effects on AD-hallmarks and synaptic
plasticity than donepezil, even in combination.

\emph{Clinical significance}

Chronic treatment with I2-IR ligands would constitute a relevant
therapeutic disease-modifying strategy against AD.

\textbf{INTRODUCTION}

Alzheimer's disease (AD) is the leading cause of dementia among the
elderly and the most common irreversible and uncurable neurodegenerative
disorder, clinically characterised by progressive behavioural
disturbances and memory loss (Murray et al., 2011).\selectlanguage{greek}\emph{Amyloid β} \selectlanguage{english}(A\selectlanguage{greek}β\selectlanguage{english})
plaques and neurofibrillary tangles consisting of hyperphosphorylated
Tau (p-Tau) are two major neuropathological AD hallmarks, which lead to
synaptic failure (Walsh and Selkoe, 2004; Selkoe, 2008; DeTure and
Dickson, 2019). Moreover, the inflammatory response triggered by A\selectlanguage{greek}β
\selectlanguage{english}deposits and Tau hyperphosphorylation, among others and mediated by
activated microglia and reactive astrocytes has a key role in the
progression of AD. (Dickson and Rogers, 1992; MERAZ RIOS et al., 2013).
Thus, targeting A\selectlanguage{greek}β \selectlanguage{english}aggregation, p-Tau, and neuroinflammation has been
proved so far, the main disease-modifying strategy for treating AD.

However, up to date, only symptomatic treatments, including
the\emph{acetylcholinesterase}inhibitors (AChEI) and the
N-methyl-D-aspartate\emph{receptor}antagonists are available for AD
therapy. Those drugs showed modest symptomatic benefits on behaviour and
cognition but they did not halt its progression(Grossberg, 2003; Mehta
et al., 2012). Among AChEI, donepezil is clinically used for cognitive
dysfunction in AD (Giacobini, 2000). Besides its main effects related to
the enhancement of cholinergic transmission, donepezil has been
demonstrated to exert the potential for disease pathway modifications in
AD, including attenuation of A\selectlanguage{greek}β \selectlanguage{english}load and anti-inflammatory properties
\emph{in vitro} and \emph{in vivo} (Kim et al., 2014). However, at a
clinical level, it lacks a curative effect, thereby the identification
of new molecular targets for the development of treatments is crucial.
In this context, to further enhance the non-cholinergic therapeutic
effects of donepezil, a combination of donepezil with other
neuroprotective agents could provide a novel approach to preserve the
cognitive function and/or delay AD pathology.

I2 imidazoline receptors (I2-IR) are receiving growing attention due to
the neuroprotective effects in the central nervous system (CNS)
(Bousquet et al., 2020). In the brain, I2-IR are found in both neurons
and glial cells (Regunathan et al., 1993; Olmos et al., 1994), and their
modulation has been associated with neurodegenerative disorders,
including AD (Ruiz et al., 1993). Most notably, the density of I2-IR was
found increased in AD patients (Garcia-Sevilla et al., 1998). Several
lines of evidence provided by our group demonstrated that selective
I2-IR ligands protected against cognitive impairment ameliorating AD
pathological features related to APP processing, Tau
hyperphosphorylation, neuroinflammation and oxidative stress (OS)
processes, using well- established AD animal models (Ab\selectlanguage{ngerman}ás et al., 2017;
Griñán-Ferré et al., 2019; Abás et al., 2020; Vasilopoulou et al.,
2020b). Likewise,\emph{agmatine}, the proposed endogenous ligand for
I2-IR, prevented cognitive deficits in A\selectlanguage{greek}β \selectlanguage{english}1-42 peptide injected mice and
of note its effect was augmented and attenuated by I2-IR agonists and
antagonists respectively (Kotagale et al., 2020). Collectively, this
evidence supports the potential therapeutic effect of I2-IR ligands in
AD.

Among the I2-IR ligands, the selective I2-IR ligand LSL60101
{[}2-(2-benzofuranyl)imidazole){]} (Ki ratio for
\selectlanguage{greek}α\selectlanguage{english}\textsubscript{2}/I2-receptors=286) has been associated with the
induction of several central effects, such as acute hyperphagic effects
(Menargues et al., 1994), inhibition of the development of
opioid-induced tolerance and potentiation of morphine analgesia (Boronat
et al., 1998). Interestingly, LSL60101 was shown to promote neuronal
protection mediated by the induction of reactive astrocytes (Casanovas
et al., 2000). However, the neuroprotective effect of LSL60101 on AD
pathological conditions has not been reported.

In the present \emph{in vivo} study, we explored the I2-IR ligand
LSL60101 beneficial effects on the behavioural capabilities and
cognitive impairments presented in AD, as well as on AD hallmarks,
including neuroinflammation, glial reactivity and synaptic plasticity by
using the 5XFAD mouse model, a widely accepted transgenic mouse model
for early-onset AD. Additionally, the comparative effect
with\emph{donepezil}, considered a symptomatic AD treatment, was
investigated alone and in combination therapy with the I2-IR ligand
LSL60101 to decipher joint effects of both compounds in ameliorating AD
pathology and molecular changes presented by 5XFAD mice.

\textbf{2. METHODS}

\textbf{2.1 Animals}

The 5XFAD mouse model is a well-characterised double transgenic
APP/PSEN1 model, which co-expresses 5 familial AD mutations. This animal
model incorporates AD pathological characteristics including early
plaque formation and gliosis starting at 2 months, robust cognitive and
behavioural deficits such as memory impairment, reduced anxiety and
social disturbances starting at 4-5 months, and neuronal loss at 6
months. (Oakley et al., 2006; Landel et al., 2014; Gri\selectlanguage{ngerman}ñán-Ferré et al.,
2018). Thus, at the selected age of 7 months, 5XFAD mice provide a
severe AD pathological landscape suitable for the evaluation of the drug
effects.

In the present study, 5XFAD (n = 47) and Wild-Type (WT, n = 46) female
mice (7-month-old) were used to perform behavioural and molecular
analyses. Females were used because AD incidence is higher in women and
few studies are available. WT animals were randomly divided into WT
Control (\textbf{WT Ct} ) (n=11), WT treated with donepezil (1
mg\textsuperscript{-1} kg\textsuperscript{-1} day\textsuperscript{-1})
(\textbf{WT Dp} ) (n=12), LSL60101 (\textbf{WT LSL} ) (1
mg\textsuperscript{-1} kg\textsuperscript{-1} day\textsuperscript{-1})
(n=12), and the co-treatment donepezil (1
mg\textsuperscript{-1}kg\textsuperscript{-1} day\textsuperscript{-1})
and LSL60101 (1 mg\textsuperscript{-1} kg\textsuperscript{-1}
day\textsuperscript{-1}) (\textbf{WT Dp+LSL} ). 5XFAD mice were randomly
divided into: 5XFAD Control (\textbf{5XFAD Ct} ) (n=11), 5XFAD treated
with Donepezil (1 mg\textsuperscript{-1} kg\textsuperscript{-1}
day\textsuperscript{-1}) (\textbf{5XFAD Dp} ) (n=12), LSL60101
(\textbf{5XFAD LSL} ) (1 mg\textsuperscript{-1} kg\textsuperscript{-1}
day\textsuperscript{-1}) (n=12), and the co-treatment Donepezil (1
mg\textsuperscript{-1}kg\textsuperscript{-1} day\textsuperscript{-1})
and LSL60101 (1 mg\textsuperscript{-1} kg\textsuperscript{-1}
day\textsuperscript{-1}(\textbf{5XFAD Dp+LSL} ). The animals had free
access to food and water and were kept under standard temperature
conditions (22 ± 2°C) and 12-h/12-h light/dark cycles (300 lux/0 lux).
Compounds were dissolved in 1.8\% (2-hydroxypropyl)-\selectlanguage{greek}β-\selectlanguage{english}cyclodextrin and
administered through drinking water for 4 weeks. Control groups received
water plus 1.8\% (2-hydroxypropyl)-\selectlanguage{greek}β-\selectlanguage{english}cyclodextrin during the treatment
period. The sample size for the intervention was chosen following
previous studies in our laboratory and using one of the available
interactive tools (\emph{http://www.biomath.info/power/index.html}).
Moreover, the animal number mismatch among experimental groups was due
to the exclusion of mice by death or ethical reasons according to the
final point indicated in the approved protocol.

After 4 weeks of treatment, behavioural and cognitive tests were
performed to study the effects of treatment on learning, memory, anxiety
behaviour and social interaction (Fig. 1a). Weight and water consumption
were controlled each week, and compounds concentrations were adjusted
accordingly to reach the optimal dose until the euthanasia. All studies
and procedures for the mouse behaviour tests, brain dissection and
extractions followed the ARRIVE (Lilley et al., 2020) and standard
ethical guidelines (European Communities Council Directive 2010/63/EU
and Guidelines for the Care and Use of Mammals in Neuroscience and
Behavioural Research, National Research Council 2003) and were approved
by Bioethical Committees from the University of Barcelona and the
Government of Catalonia.

\textbf{2.2 Behavioural tests}

\textbf{2.2.1 Novel Object Recognition Test (NORT)}

A modification of NORT protocol was performed (Ennaceur and Delacour,
1988). In brief, mice were placed in a 90\selectlanguage{ngerman}° two-arm (25 x 20 x 5 cm)
black maze, with removable walls for easy cleaning and light intensity
in mid-field was 30 lux. Before the memory, trials mice were habituated
to the apparatus for 10 min for 3 days. On day 4, the animals were
submitted to a 10 min acquisition trial, in which they were allowed to
freely explore two identical objects located at the end of each arm
(First trial-Familiarization). After 2h (for short-term memory
evaluation) and 24h (for long-term memory evaluation) from the first
trial, the mice were submitted to a 10 min retention trial, in which one
of the two identical objects had been replaced by a novel one. The
behaviour was recorded, and the time spent that the mice spent exploring
the new object (TN) and the old one (TO) were measured manually.
Exploration was defined as sniffing or touching the objects with nose
and/or forepaws. The discrimination index (DI) was calculated as
(TN-TO)/(TN+TO). To avoid object preference biases, objects were
alternated. 70\% EtOH was used to clean the arms and objects after each
trial for the elimination of olfactory cues.

\par\null

\textbf{2.2.2 Morris Water Maze (MWM)}

The MWM test was performed as described previously (Griñan-Ferré et al.,
2016) in an open circular pool, filled with water which temperature was
maintained at 22 °C ± 1. The water surface was divided into four
quadrants (Q1, Q2, Q3, and Q4) by two principal perpendicular axes, and
five starting points were set (1,2,3,4). Four visual clues were placed
on the walls of the tank (1,2,3,4). The animals' swimming paths were
recorded, and the data were analysed with SMART version 3.0 software. On
day 1, mice were placed individually into the pool, facing the wall, and
allowed to swim for 60 seconds in order to be habituated to the
experimental conditions. On day 2, a white platform was submerged 1.5 cm
below the water level in the middle of the Q1 platform and the
acquisition phase took place for 5 days. Each day the animals were
submitted to five trials starting from the positions set in random
order. At each trial mice were allowed to swim for 60 seconds and if not
able find the platform within 60 seconds were guided to the visible
platform. The mice remained for 30s onto the platform for spatial
orientation. There was no resting phase between each trial and the
subsequent one. 24h after the last training, a memory test was performed
after the platform was removed from the pool in which the mice were
tested for 60s. The distance to target, and the time spent in platform
quadrant (Q1), among other parameters, were measured.

\textbf{2.2.3 Open Field (OF)}

Emotional alterations and locomotor activity were evaluated by the OF
test using a white plywood apparatus (50 × 50 × 25 cm) as previously
described (Archer, 1973; Griñan-Ferré et al., 2016).The apparatus'
ground was divided into the center and peripheral area. Each individual
was placed at the centre of the open field and allowed to explore the
apparatus for 5 min. The apparatus was cleaned with 70\% ethanol after
between trials. The behaviour was recorded and later analysed with SMART
ver. 3.0 software (Panlab). The locomotor activity of the mice
calculated as the sum of total distance travelled in 5 min, the centre
stay duration and the number of rearings were evaluated.

\textbf{2.2.4 Elevated Plus Maze (EPM)}

Animals were tested for anxiety-like behaviour by performing the EPM
test, based on a previously described protocol (Walf and Frye, 2007).
The EPM apparatus consisted of two open arms (30 x 5 x 15 cm) and two
closed arms (30 x 5 x 15 cm). The mice were placed at the junction of
the arms and allowed to explore the apparatus for 5 min freely. EPM
apparatus was cleaned with 70\% ethanol between tests. The behaviour was
recorded and later analysed with SMART ver. 3.0 software (Panlab).
Parameters recorded included the total distance travelled during the 5
min test, the time spent in open arms, closed arms, and centre, as well
as the number of rearings.

\textbf{2.2.5 Three-Chamber Test (TCT)}

Social behaviour of the mice was evaluated by the TCT following a
previously described protocol (Companys-Alemany et al., 2020). A box
(15x15x20 com) divided into three equally dimensioned rooms with
openings among them was used. The mice were submitted to 15-min trials.
First, each mouse was placed in the centre of the box and allowed to
explore the three chambers for 5 min (habituation). The entries to each
room were measured manually. Afterwards, an intruder (same-sex and age)
was placed in a metal cage at one of the rooms, and behaviour was
recorded for 10 min. The time spent in each room and the time
interacting with the intruder (e.g., sniffing, grooming) were measured
manually. The TCT apparatus was cleaned with 70\% ethanol between the
trials to eliminate olfactory cues.

\textbf{2.3 Brain processing}

Mice were euthanised by cervical dislocation 3 days after the
behavioural and cognitive tests were completed. The brains were
immediately removed from the skulls, and the hippocampi were dissected,
frozen and maintained at \selectlanguage{english}-80degC. For immunohistochemistry (IHC)
experiments, mice were anesthetised (ketamine 100
mg\textsuperscript{-1}kg\textsuperscript{-1} and xylazine 10
mg\textsuperscript{-1}kg\textsuperscript{-1}), intraperitoneally and
then perfused with 4\% paraformaldehyde (PFA) diluted in 0.1 M phosphate
buffer solution intracardially. Their brains were removed and postfixed
in 4\% PFA overnight at 4degC. Afterwards, the solutions were changed to
PFA + 15\% sucrose. Finally, the brains were frozen on powdered dry ice
and stored at -80degC until sectioning.

\textbf{2.4 Protein levels determination by Western blotting}

For protein extraction, tissue samples were homogenised in lysis buffer
containing phosphatase and protease inhibitors (Cocktail II,
Sigma-Aldrich). Total protein levels were obtained, and protein
concentration was determined by the method of Bradford. For WB, aliquots
of 15 \selectlanguage{greek}μ\selectlanguage{english}g of hippocampal protein were used. Protein samples were
separated by Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis
(SDS-PAGE) (8-16\%) and transferred onto Polyvinylidene difluoride
(PVDF) membranes (Millipore). Afterwards, membranes were blocked in 5\%
bovine serum albumin (BSA) in 0,1\% Tris-buffered saline - Tween20
(TBS-T) for 1h at room temperature, followed by overnight incubation at
4\selectlanguage{ngerman}°C with the primary antibodies listed in Supplementary Table S1.
Membranes were washed and incubated with secondary antibodies for 1h at
room temperature. Immunoreactive proteins were viewed with a
chemiluminescence-based detection kit, following the manufacturer's
protocol (ECL Kit; Millipore) and digital images were acquired using a
ChemiDoc XRS+ System (BioRad). Semi-quantitative analyses were carried
out using ImageLab software (BioRad), and results were expressed in
Arbitrary Units (AU), considering control protein levels as 100\%.
Protein loading was routinely monitored by immunodetection of
glyceraldehyde-3-phosphate dehydrogenase (GADPH) or \selectlanguage{greek}β-\selectlanguage{english}actin.

\textbf{2.5 RNA extraction and gene expression determination}

Total RNA isolation from hippocampal samples was performed using the
TRIzol\selectlanguage{ngerman}® reagent according to the manufacturer's instructions (Bioline
Reagent). The yield, purity and quality of RNA were determined
spectrophotometrically with a NanoDrop\selectlanguage{english}ND-1000 apparatus (Thermo
Scientific) and an Agilent 2100B Bioanalyzer (Agilent Technologies). RNA
samples with 260/280 ratios and RINs higher than 1.9 and 7.5,
respectively, were selected. Reverse Transcription-Polymerase Chain
Reaction (RT-PCR) was performed. Briefly, 2 \selectlanguage{greek}μ\selectlanguage{english}g of messenger RNA (mRNA)
was reverse transcribed using a high-capacity cDNA reverse transcription
kit (Applied Biosystems).

SYBR\selectlanguage{ngerman}® Green real-time PCR was performed using a Step One Plus Detection
System (Applied-Biosystems) with SYBR® Green PCR Master Mix
(Applied-Biosystems). Each reaction mixture contained 6.75 \selectlanguage{greek}μ\selectlanguage{english}L of
complementary DNA (cDNA) (with a concentration of 2\selectlanguage{greek}μ\selectlanguage{english}g), 0.75 \selectlanguage{greek}μ\selectlanguage{english}L of each
primer (with a concentration of 100nM), and 6.75 \selectlanguage{greek}μ\selectlanguage{english}L of SYBR\selectlanguage{ngerman}® Green PCR
Master Mix (2x).

The data were analysed utilising the comparative cycle threshold (Ct)
(\selectlanguage{greek}ΔΔ\selectlanguage{english}Ct) method, in which the levels of a housekeeping gene are used to
normalize differences in sample loading and preparation. Normalization
of expression levels was performed with \selectlanguage{greek}β-\selectlanguage{english}actin. The primer sequences
and TaqMan probes used in this study are presented in Supplementary
Table S2. Each sample was analyzed in duplicate, and the results
represent the n-fold difference in the transcript levels among different
groups.

\textbf{2.6. Glial immunohistochemical identification}

Brain coronal sections of 30 \selectlanguage{greek}μ\selectlanguage{english}m were obtained (Leica Microsystems CM
3050S cryostat, Wetzlar, Germany) and kept in a cryoprotectant solution
at -20\selectlanguage{ngerman}°C until use. Free-floating slices were placed in a 24-well plate
and washed with 0.01M PBS. Next, the free-floating sections were blocked
with 0.1M PBS solution containing 1\% BSA, 0,3\% Triton X-100 for 20min
at room temperature. Afterwards, slices were washed with PBS 0.01M two
times for 5 min each and were incubated with the primary antibodies
listed in~Table X overnight at 4°C. The primary antibodies were diluted
in a 0.1M PBS solution containing 1\% BSA and 0.3\% Triton x-100. On the
following day, the coronal slices were washed with 0.1M PBS 0.1M 2 times
for 5 min each and then incubated with the secondary antibodies listed
in Supplementary Table S1 at room temperature for 1h. Later, the
sections were washed 2 times for 5 min each with 0.1M PBS and were
incubated with 5\selectlanguage{greek}μ\selectlanguage{english}M Hoechst staining solution (Sigma-Aldrich, St. Louis,
MO) for 5 min in the dark at room temperature. After being washed, the
slices were mounted using Fluoromount-G (EMS, USA).

\selectlanguage{greek}\textbf{2.7. Amyloid β plaques histology}\selectlanguage{english}

A\selectlanguage{greek}β \selectlanguage{english}plaques were stained with Thioflavin-S. Brain coronal sections of 30
\selectlanguage{greek}μ\selectlanguage{english}m were obtained (Leica Microsystems CM 3050S cryostat, Wetzlar,
Germany) and kept in a cryoprotectant solution at -20\selectlanguage{ngerman}°C until use.
Free-floating slices were placed in a 24-well plate and washed with
0.01M PBS for 5 min at room temperature to be rehydrated. Next, the
brain sections were washed with 70\% ethanol for 1 min followed by a
wash with 80\% ethanol for 1 min. The slices were then incubated with
0.3\% Thioflavin-S (Sigma-Aldrich) solution for 15 min at room
temperature in the dark. Afterwards, the samples were washed using 80\%,
70\% and 50\% EtOH for 1 min each. Three 2-min washes with 0.1M PBS and
the slices were mounted using Fluoromount-G (EMS, USA).

\textbf{2.8 Image acquisition and analysis}

Image acquisition was performed with a fluorescence laser microscope
(Olympus BX51, Germany) using 4X, 10X, 20X objectives, and images were
analysed using Imaje J software as previously described. For
quantification of amyloid plaques similar and comparable histological
areas were selected, focusing on the adjacent positioning of the whole
cortical area and the hippocampus. The images were converted to 8-bit
grayscale images, thresholder within the linear range and the number of
particles (Analyse particle function 10-Infinity), as well as the
percentage of area covered by Thioflavin-S (20X objective), was
calculated and averaged from two different measurements per animal.
Glial fibrillary acidic protein (GFAP) and Iba-1 stained images (10X)
were acquired, maintaining constant exposure for all samples across
single experiments. The fluorescence intensity of the positive cells was
measured in DG, CA1 and CA3 areas of the hippocampus. Quantification was
averaged from two to three different measurements.

\textbf{2.9 Statistical Analysis}

The data and statistical analysis comply with the recommendations of the
British Journal of Pharmacology on experimental design and analysis in
pharmacology (Curtis et al., 2018). Group size may vary according to
power analysis and expertise of the authors regarding the behavioural
tests (Griñan-Ferré et al., 2016; Griñán-Ferré et al., 2018) and
statistical analysis was undertaken only for studies where each group
size was at least n=5. Blinded analysis was performed for behavioural
test.All data are expressed as the mean ± standard error of the mean
(SEM). Statistical analysis was conducted using GraphPad Prism version 8
statistical software. All data were tested for normal distribution and
equal variance. In the cognitive and behavioural studies, means were
compared with two-way ANOVA. In molecular studies, means were compared
with two-tailed Student's t-test (WT Control vs 5XFAD Control) or
one-way ANOVA followed by Tukey's post hoc tests (5XFAD Control vs 5XFAD
treated groups). Statistical significance was considered when P values
were \textless{}0.05. Statistical outliers were determined with Grubbs'
test and when necessary were removed.

\textbf{2.10 Nomenclature of Targets and Ligands}

Key protein targets and ligands in this article are hyperlinked to
corresponding entries in http://www.guidetopharmacology.org, the common
portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY and are
permanently archived in the Concise Guide to PHARMACOLOGY 2019/20.

\textbf{RESULTS}

\textbf{I2-IR ligand LSL60101 and donepezil improve memory deficits in
5XFAD mice.}

Short- and long-term working memory were evaluated by NORT. 7-month-old
5XFAD mice presented robust cognitive deficits when compared to WT.
LSL60101 treatment resulted in a rapid and sustained recovery of
cognitive function by increasing the DI in both 2h and 24h memory tests
(Figure 1b-c). Donepezil enhanced but did not sustain memory function in
5XFAD mice, as a significant increase of the DI was found after 2h, but
not at 24h memory test (Figure 1c). Co-treatment did not improve
cognition in comparison with individual treatments (Figure 1b-c).
Treatments had no significant effects on WTs cognitive performance
(Figures 1b-c).

For spatial learning and memory evaluation, the MWM was performed. After
5 days of training, all experimental groups presented curves with
progressively shorter path length on consecutive days. Of note, the path
length to the platform was significantly decreased in LSL60101 treated
5XFAD mice when compared to 5XFAD controls (Figure 1d). In the probe
trial, 5XFAD mice showed a decreased percentage of time spent in the
platform quadrant while the mice spent significantly more time in the
quadrant opposite to the platform (Figure 1e,1h). LSL60101 treatment
significantly increased the time spent in the platform quadrant in the
5XFAD treated mice when compared to both vehicle and donepezil treated
5XFAD, whereas LSL60101 treatment had no effect on WTs (Figures 1e,
1f,1h). Neither donepezil nor co-treatment improved 5XFAD mice spatial
memory (Figures 1d, 1e). Although WT treated mice performed better
(Figures 1d,1f,1h). All treatments decreased the path length to the
platform albeit not significantly, due to different performance of
individual mice (Figure 1g).

\textbf{I2-IR ligand LSL60101 does not affect behavioural and emotional
disturbances in 5XFAD mice in contrast to donepezil.}

We also investigated the effect of the treatments on the 5XFAD and WT
mice anxiety-like behaviour by performing the OF and EPM tests. No
differences in locomotor activity were observed among the WT and 5XFAD
groups (Figure 2a). 5XFAD mice presented a significant increase in the
time spent in the centre of the open field compared to WT mice (Figure
2b). No effect was observed on the WT mice behaviour after treatments.
5XFAD treated with donepezil but not with LSL60101 showed a significant
decrease in the time spent in the centre compared to 5XFAD controls,
reverting to the WT healthy phenotype (Figure 2b). Co-treatment
LSL60101/donepezil displayed the same results that showed donepezil
alone, in all parameters evaluated (Figure 2b and Supplementary table
S3). Similarly, in the EPM, 5XFAD mice spent significantly more time in
the open arms and less in the closed arms in comparison with age-matched
WTs (Figures 2d, 2e). Donepezil had a positive effect by reverting the
5XFAD EPM parameters evaluated to those showed by WT group (Figures
2d-f, Supplementary table S4). I2-IR ligand treatment alone did not
affect any of the EPM parameters studied, whereas co-treatment
maintained the donepezil values. Treatments did not induce significant
changes in EPM parameters evaluated in WT mice (Supplementary Table S4).

\textbf{3.3 I2-IR ligand LSL60101 and donepezil ameliorate social
deficits presented by 5XFAD mice.}

To evaluate the effect of treatments on social behaviour, the mice were
subjected to the TCT. No differences in the number of the entries to
each chamber were determined in the habituation phase in any tested
group (Figure 2g). In contrary mice spent more time in the intruder's
chamber during the test phase in all experimental conditions (Figure
2h). When the social interaction was evaluated, 5XFAD mice spent
significantly less time interacting with the intruder when compared to
WT healthy control. All treatments improved social impairments in 5XFAD
treated groups by increasing the time of interaction compared to the
5XFAD controls, but only 5XFAD treated with donepezil reached
significance (Figure 2i), whereas for LSL60101 p\textless{}0.06 was
calculated. WT treated mice presented no differences compared to WT
controls.

\selectlanguage{greek}\textbf{3.4 I2-IR ligand LSL60101, but not donepezil, reduces Aβ
plaques. By contrast, donepezil/LSL60101 attenuate Aβ pathology in 5XFAD
mice.}\selectlanguage{english}

The number of amyloid plaques in 5XFAD mice was assessed by
histochemical staining with Thioflavin-S. Following treatment with
LSL60101, there was a significant decrease in the total number and area
of the plaques in 5XFAD mice when compared to the 5XFAD controls,
demonstrating a neuroprotective function of I2-IR ligand regarding the
senile plaque formation. 4-week-treatment with donepezil did not reduce
the amyloid deposition significantly in 5XFAD mice (Figure 3a-3c). The
protein levels of A\selectlanguage{greek}β \selectlanguage{english}determined by WB tended to decrease in all treated
groups without reaching significance (Figure 3d, 3f). Interestingly,
alterations in the levels of proteins implicated in the APP processing
showed complementary results in the combination of LSL60101 and
donepezil treatment. In this line, the protein levels of C-terminal
fragments (CTFs) were found significantly reduced in LSL60101 treated
groups when compared to the 5XFAD controls while donepezil/LSL60101
treated group showed a higher decrease in CTFs compared with monotherapy
(Figure 4a, 4g). The protein levels of phosphorylated amyloid precursor
protein (p-APP) at Th668 were significantly decreased only for the
combination of donepezil/LSL60101 treated animals (Figure 4b). Soluble
APP\selectlanguage{greek}β \selectlanguage{english}(sAPP\selectlanguage{greek}β\selectlanguage{english}) levels were found significantly decreased in
donepezil/LSL60101 treated group, whereas soluble APP\selectlanguage{greek}α \selectlanguage{english}(sAPP\selectlanguage{greek}α\selectlanguage{english}) levels
were found significantly increased after combination treatment when
compared to 5XFAD controls or donepezil treated mice (Figure 4c, 4d,
4g). No differences in the gene expression of \emph{APP} were found
among the groups (Figure 4h). Regarding the levels of
BACE1\selectlanguage{greek}\emph{(β-secretase}\selectlanguage{english}) and ADAM10 (\selectlanguage{greek}α-\selectlanguage{english}secretase), no significant
differences were observed among the 5XFAD groups (Figures 4e-4g).
However, when the gene expression and protein levels of enzymes
implicated in amyloid degradation were studied, donepezil/LSL60101
treatment slightly increased gene expression of \emph{insulin-degrading
enzyme} (\emph{Ide} ) and \emph{neprilysin}(\emph{Nep} ) (Figure
4i)\emph{.}

\textbf{3.5 I2-IR ligand LSL60101 and combination with donepezil reduce
Tau hyperphosphorylation in 5XFAD mice}

Tau hyperphosphorylation, another major hallmark of AD, was evaluated in
the hippocampus of the 5XFAD mice. I2-IR ligand LSL60101 and the
co-treatment donepezil/LSL60101 decreased the Tau phosphorylation at the
Ser404 and Ser396, diminutions that reached significance for the
donepezil/LSL60101 treated 5XFAD mice. Of note, significant differences
in p-Tau levels were found as well between the donepezil/LSL60101 5XFAD
treated mice, and the donepezil or LSL60101 treated ones (Figure 3e,
3f).

\textbf{3.6 Effects of LSL60101 on microglia activation and inflammatory
markers expression.}

In the AD brain, the formation of A\selectlanguage{greek}β \selectlanguage{english}plaques leads to the activation of
astrocytes and reactive gliosis. To examine changes in microglia
reactivity, Iba-1 was determined by IHC experiments. Importantly,
LSL60101 treatment resulted in a significant decrease in Iba-1 levels in
the hippocampus of 5XFAD mice whereas donepezil did not affect Iba-1
immunoreactivity (Figure 5a-5d). Donepezil/LSL60101 combination did not
modify the beneficial effect of LSL60101 by itself on microglia
activation.

The gene expression of different inflammatory mediators was evaluated in
the hippocampus of 5XFAD mice. In whole, 5XFAD mice presented an evident
exacerbation of inflammatory response compared to WT mice, whereas
treatments led to the upregulation of specific markers studied.
Interestingly, significant increases or tendency to increase were
determined in\selectlanguage{greek}\emph{Interleukin 1β (Il-1b),Interleukin 6 (Il-6),}\selectlanguage{english}
and\emph{Chemokine (C-C motif) ligand 12 (Ccl12)} markers in LSL60101
treated group, while donepezil alone had no significant effect on these
markers (Figure 5e). Moreover, the gene expression of \emph{Triggering
receptor expressed on myeloid cells 2~(Trem2} ) was increased
significantly after treatment with the I2-IR ligand LSL60101 (Figure
5f).

\textbf{3.6 LSL60101 effects on astroglial activation and synaptic
dysfunction.}

All treatments were able to attenuate astrogliosis in the hippocampus of
5XFAD brains by decreasing GFAP immunoreactivity in DG, CA1 and CA3
areas in 5XFAD mice groups in comparison with untreated mice (Figures
6a-d). Likewise, synaptic plasticity markers were evaluated by WB.
Decreases in the protein levels of postsynaptic density protein 95
(PSD95) and synaptophysin (SYN) were determined in 5XFAD mice when
compared to WT mice (Figures 6f-6h). I2-IR ligand LSL60101 increased
PSD95 levels when compared to 5XFAD control (Figure 6f, 6h). SYN levels
were found increased in LSL60101, and donepezil/LSL60101 treated 5XFAD
mice, reaching significance only for the combination-treated group.
Donepezil treatment was not able to significantly modify these markers
(Figure 6g, 6h).

\textbf{DISCUSSION}

The identification of new targets for AD treatment is required due to
the lack of effective disease treatment. At present, AChEI are one of
the standard therapeutic options clinically available for AD patients;
however, those treatments provide only symptomatic benefit in AD
(Sinforiani et al., 2003; Rosini et al., 2014). Fortunately, the number
of disease-modifying drugs targeting AD hallmarks such as aducanumab
(BIIB037), which is currently in phase 3 trials, is increasing (Cummings
et al., 2020). Combination therapies of symptomatic and
disease-modifying drugs have centred attention due to the multifactorial
origin of the disease, and most current clinical trials combine
donepezil with novel neuroprotective drugs (Fr\selectlanguage{ngerman}ölich et al., 2019).
However, it remains a challenge that must be addressed to unveil new
strategies that could be more effective in disease-modifying treatment,
rather than address symptoms (Schmitt et al., 2004).

Several studies have described the symptomatic effects of donepezil in
animal models of dementia and AD, but few \emph{in vivo} studies have
evaluated donepezil neuroprotective effects regarding the
disease-modifying actions of this compound alone or in combination
(Jiangbo and Liyun, 2018; Krishna et al., 2020; Yang et al., 2020;
Ongnok et al., 2021). Here, we studied the effect of chronic low doses
of an I2IR ligand, donepezil and their combination.

In the light of our studies, we demonstrated for the first time the
neuroprotective effects of selective I2-IR ligands in the
senescence-accelerated mouse prone 8 (SAMP8), a mouse model of
late-onset AD (Griñán-Ferré et al., 2019). LSL60101, a selective I2-IR
ligand has been shown to induce several biological effects associated
with I2-IR occupancy and most importantly neuroprotective effects in the
CNS (Menargues et al 1994; Boronat et al., 1998; Casanovas et al., 2000;
Sánchez-Blázquez et al., 2000).Therefore, it represents a suitable drug
candidate to validate this receptor as a target for AD. Here, we
demonstrated the efficacy of chronic low-dose I2-IR ligand LSL60101
treatment in comparison with donepezil by assessing the beneficial
outcomes in a model of familial AD.

Cognitive abilities are the essential indicators to unveil
pharmacological effects in AD.~Firstly, chronic low-dose treatment with
the I2-IR ligand LSL60101 or donepezil reversed the cognitive deficits
presented by 7-month-old 5XFAD mice, without affecting WT mice in the
NORT paradigm. However, in the spatial memory test, only LSL60101 showed
improvements in memory. Likewise, 5XFAD exhibited improved social
behaviour after LSL60101 or donepezil treatment. In agreement with these
results, donepezil has been shown to improve social interactions in
scopolamine-induced memory impairments in mice (Riedel et al., 2009) and
in drug-trials for AD (Boada-Rovira et al., 2004). Nevertheless, the
beneficial effect of an I2-IR ligand treatment on social interaction
deficits has not been described previously.

By contrast, I2-IR ligand LSL60101 did not modify anxiety-like
behaviour, albeit previous studies have shown the \emph{in
vivo}anxiolytic and anti-depressant-like effects induced by I2-IR
ligands (Finn et al., 2003; Tonello et al., 2012). Interestingly, the
absence of anti-depressant effect after treatment with LSL60101 in
healthy rats was recently described (Hernández-Hernández et al., 2020)
further supporting our results, since anxiety-like and depressive-like
behaviour are strongly associated, sharing common molecular pathways
(Gatt et al., 2009). In contrast, and according to literature
(Fitzgerald et al., 2020), chronic treatment with donepezil showed
beneficial effects on the anxiety-related disturbances exhibited by
5XFAD mice .

Recently, we reported that the amyloidogenic APP processing pathway was
suppressed in SAMP8 and 5XFAD mice after treatment with novel I2-IR
ligands, anticipating the role of I2-IR modulation in the A\selectlanguage{greek}β \selectlanguage{english}biogenesis
(Gri\selectlanguage{ngerman}ñán-Ferré et al., 2019; Abás et al., 2020; Vasilopoulou et al.,
2020b). Accordingly, in this study, we also demonstrated that chronic
low-dose treatment with I2-IR ligand LSL60101 attenuated the amyloid
plaque burden in 5XFAD mice. In addition, A\selectlanguage{greek}β \selectlanguage{english}plaques reduction was
accompanied by a decrease in CTFs and A\selectlanguage{greek}β \selectlanguage{english}hippocampal protein levels, as
well as favourable modifications in APP processing after treatment.
Conversely, recently it was reported that the I2-IR ligand BU224 does
not ameliorate A\selectlanguage{greek}β \selectlanguage{english}amyloidosis in 5XFAD mice (Mirzaei et al., 2020), but
improves memory. In contrast with LSL60101 like molecules, BU224 blocked
the memory-enhancing effect of agmatine in memory deficits induced by
A\selectlanguage{greek}β\selectlanguage{english}\textsubscript{1-42} in mice (Kotagale et al., 2020). These
discrepancies between I2-IR ligands can be explained by differences in
compound administration conditions such as dose, time
(sub-chronic\emph{vs.} chronic) and route of administration. Thus, we
hypothesise that low doses of LSL60101, as well as the chronicity of
treatment, has a clear beneficial effect on amyloid burden because of
differential characteristics among I2-IR ligands (S\selectlanguage{ngerman}ánchez-Blázquez et
al., 2000; Garau et al., 2013).

Several studies have demonstrated the effect of donepezil on A\selectlanguage{greek}β
\selectlanguage{english}pathology in AD models, reporting either beneficial changes (Dong et
al., 2009; Takada-Takatori et al., 2019) or lack of effect (Ju and Tam,
2020). Here, low-dose donepezil treatment did not induce significant
changes on neither A\selectlanguage{greek}β \selectlanguage{english}plaques nor APP processing in 7-month-old 5XFAD.
Of note, co-administration of I2-IR ligand LSL60101 with donepezil was
shown to have a greater effect on APP processing, as the co-treatment
donepezil/LSL60101 induced an increase in A\selectlanguage{greek}β \selectlanguage{english}degradation enzymes gene
expression in 5XFAD mice, which was not determined in the other treated
groups. To sum up, this is the first time that an I2-IR ligand was shown
to be effective in reducing A\selectlanguage{greek}β \selectlanguage{english}burden in \emph{in vivo} mice model of
AD.

Presence of p-Tau, another major AD hallmark, in the 5XFAD model is
supported by previous studies suggesting that Tau pathology may be
downstream from A\selectlanguage{greek}β \selectlanguage{english}pathology (Blanchard et al., 2003; Saul et al.,
2013). I2-IR ligand LSL60101 ameliorated Tau pathology in the
hippocampus of 5XFAD mice. Interestingly, it was shown recently that
chronic treatment with idazoxan, a mixed \selectlanguage{greek}α\selectlanguage{english}2/I2 ligand, reduced p-Tau
reversing cognitive deficits in AD mice, because of its \selectlanguage{greek}α\selectlanguage{english}2 blockade
action (Zhang et al., 2020). In this case, the effect of LSL60101 on Tau
pathology can be attributed to its I2 selectivity, more than to the \selectlanguage{greek}α\selectlanguage{english}2
one. Surprisingly, the p-Tau reduction reached significance in the 5XFAD
mice treated with the combination of LSL60101 with donepezil
demonstrating, in this case, a putative additive effect of the drugs on
Tau pathology. Indeed, amelioration of Tau pathology has been induced in
AD animal models both by donepezil (Yoshiyama et al., 2010) and by I2-IR
ligand treatments (Gri\selectlanguage{ngerman}ñán-Ferré et al., 2019; Vasilopoulou et al.,
2020b). It is possible that the activation of distinct molecular
pathways by the two molecules with different modes of actions resulted
in a remarkable p-Tau reduction observed in the donepezil/LSL60101
treated mice group.

It is well-established that A\selectlanguage{greek}β \selectlanguage{english}accumulation jointly with p-Tau increases
microglial activation and inflammatory mediators' production in AD
brains (Akiyama et al., 2000; Serrano-Pozo et al., 2011; Zhang and
Jiang, 2015). On the one hand, chronic low-dose LSL60101 treatment
reduced microgliosis in 5XFAD mice in contrast to the standard of care
donepezil, explaining the decrease in the amyloid deposition that in
turn would lead to a decrease in gliotic response after LSL60101
treatment. On the other hand, inflammatory gene expression increase
(\selectlanguage{greek}\emph{Il-1β, Il-6,}\selectlanguage{english} and \emph{Ccl12} ) was observed after treatment
with I2-IR ligand LSL60101 but not with donepezil. Interestingly, this
is further supported by a significant upregulation of \emph{Trem2} gene
expression determined in the LSL60101 treated mice, further confirming
the neuroinflammatory modulation by I2-IR ligand LSL60101 (Hwang et al.,
2010; Gri\selectlanguage{ngerman}ñán-Ferré et al., 2019; Vasilopoulou et al., 2020a).
Furthermore, increased \emph{Trem2} expression has been shown to
reprogram microglia responsivity mediating microglial cytokine release,
migration and clearance of A\selectlanguage{greek}β \selectlanguage{english}deposits, ameliorating neuropathological
and behavioural deficits of AD mouse models (Lee et al., 2018; Zhao et
al., 2018).

It has been described that the I2-IR modulate the expression of
astrocyte marker GFAP, especially considering their primary location in
astrocytes (Regunathan et al., 1993; Olmos et al., 1994). GFAP
diminution was observed in SAMP8 after chronic treatment with selective
I2-IR ligands (Gri\selectlanguage{ngerman}ñán-Ferré et al., 2019; Vasilopoulou et al., 2020b).
In agreement with those results, chronic low-dose treatment with I2-IR
ligand LSL60101 attenuated astrogliosis in 5XFAD mice. By contrast, it
has been shown that chronic treatment with LSL60101 increased GFAP
immunoreactivity (Alemany et al., 1995) resulting in reactive
astrocytosis and preventing motoneuron cell death in neonatal rats
(Casanovas et al., 2000). However, here, in a neurodegenerative
landscape provided by the 5XFAD model, the diminution of GFAP reactivity
ran in parallel with the attenuation of the A\selectlanguage{greek}β \selectlanguage{english}pathology and microglial
activation observed after LSL60101 and donepezil treatment, given
further support to the beneficial effects of I2-IR ligand on mice
behaviour. Ultimately, we demonstrated that chronic low-dose treatment
with I2-IR ligand as well as donepezil enhanced synaptic plasticity,
further supporting the cognitive and behavioural improvement induced by
the LSL60101 in 5XFAD mice.

\textbf{CONCLUSIONS}

Collectively, we report that chronic low-dose treatment with I2-IR
ligand LSL60101 reversed cognitive deficits in 5XFAD mice, changing AD
neuropathological hallmarks, including glial activation and synaptic
dysfunction. Strikingly, treatment with I2-IR ligand LSL60101 was found
to exert more significant beneficial effects under neurodegenerative
process caused by A\selectlanguage{greek}β \selectlanguage{english}pathology than donepezil, However, combination
treatment only showed discrete synergistic effects at the molecular
level (e.g., tau hyperphosphorylation or synaptic plasticity),
suggesting that increased dosage and/or duration of the treatment may be
able to produce better effects on both behaviour and AD-hallmarks,
targeting simultaneously pathological and symptomatic reliefs. In
conclusion, our findings demonstrate the therapeutic potential of the
I2-IR for AD treatment as a disease-modifying single therapy and provide
new insights for their efficacy.

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\textbf{Figure 1. Effects of low-dose chronic treatment with I2-IR
LSL60101, Donepezil and co-administration on cognitive status in 5XFAD
and WT mice. (a)} Scheme of experimental design. \textbf{Results of
NORT:} Discrimination index calculated by using exploration time for
novel and familiar object \textbf{(b)} in the short-term term memory
test session (2h) (WT, n=10-12 per group; 5XFAD n=11-12 per
group)\textbf{(c)} in the long-term memory test session (24h) (WT,
n=10-12 per group; 5XFAD n=10-12 per group). \textbf{Results of MWM} :
\textbf{(d)}Distance to target (platform) (cm) during the training
session (WT n=10-11 per group; 5XFAD n=10-12 per group) \textbf{(e)}
Quadrant preference in the test session as time (\%) spent in each
quadrant WT n=9-12 per group; 5XFAD n=10-12 per group) \textbf{(f)} Time
(\%) spent in platform quadrant in the test session (WT n=9-12 per
group; 5XFAD n= 10-12 per group) \textbf{(g)} Distance to target
(platform) (cm) in the test session (WT n=9-12 per group; 5XFAD n=10-12
per group) \textbf{(h)}Representative images of trajectory during memory
test. Bars represent mean\selectlanguage{ngerman}±SEM. one-way and two-way ANOVA with Tukey post
hoc analysis, *P\textless{}0.05.

\textbf{Figure 2. Effects of low-dose chronic treatments I2-IR LSL60101,
Donepezil and co-administration on behavioral and social status in 5XFAD
mice and WT controls. Results of OF: (a)} Locomotor activity measured as
distance travelled (cm) WT n=10-12 per group; 5XFAD n=11-12 per
group\textbf{(b)} Time spent in the center (sec) (WT n=10-12 per group;
5XFAD n=9-11 per group) \textbf{(c)} number of rearings (WT n=11-12 per
group; 5XFAD n=10-12 per group). \textbf{Results of EPM: (d)} Time (\%)
spent in Open Arms (WT n=11-12 per group; 5XFAD n=10-12 per
group)\textbf{(e)} Time (\%) spent in closed arms (WT n=11-12 per group;
5XFAD n=10-12 per group) \textbf{(f)} number of rearings (WT n= 11-12
per group; 5XFAD n=11-12 per group). \textbf{Results of TCT: (g)}
Entries in chambers (A1, A2) in the habituation phase (n) (WT n=11-12
per group; 5XFAD n=11-12 per group) \textbf{(h)} Time in chambers
(Empty, Stranger) during the test session (WT n=10-11 per group; 5XFAD
n=9-11 per group)\textbf{(i)} time of interaction with intruder (sec) in
the test session (WT n=11-12 per group; 5XFAD n=10-11 per group). Bars
represent mean±SEM. Student's t-test or two-way ANOVA with Tukey post
hoc analysis, *P\textless{}0.05;

\textbf{Figure 3. Effects of low dose chronic treatments I2-IR LSL60101,
donepezil and co-administration on AD hallmarks in 5XFAD mice.
(a)}Histological images of amyloid plaques stained with Thioflavin-S and
quantification of \textbf{(b)} number of the plaques in cortex and
hippocampus (\textbf{c} ) and area (\%) covered by plaques in the DG
area of the hippocampus in the 5XFAD mice. Representative Western blot
and quantifications \textbf{(d-f)} for \selectlanguage{greek}β-\selectlanguage{english}Amyloid p-Tau(Ser404) and
p-Tau(ser396) in the hippocampus of 5XFAD mice. Values in bar graphs are
adjusted to 100\% for protein levels of the control WT control or the
control 5XFAD. Bars represent mean\selectlanguage{ngerman}±SEM. Student's t-test or one-way
ANOVA with Tukey post hoc analysis, *P\textless{}0.05; n=5-6 per group.

\selectlanguage{greek}\textbf{Figure 4. Effects of low dose chronic treatments I2-IR LSL60101,
donepezil and co-administration on Aβ pathology and APP processing in
5XFAD mice.}\selectlanguage{english} Representative Western Blots and Quantifications
for\textbf{(a-g)} CTFs, p-APP, sAPP\selectlanguage{greek}β, \selectlanguage{english}sAPP\selectlanguage{greek}α, \selectlanguage{english}BACE 1, ADAM10 in the
hippocampus of 5XFAD mice. Values in bar graphs are adjusted to 100\%
for protein levels of the control WT control or the control 5XFAD.
Representative gene expression in the hippocampus of the 5XFAD mice
for\textbf{(h)} \emph{APP} and \textbf{(i)} \emph{IDE, Nep.} Gene
expression levels were determined by real-time PCR. Bars represent
mean\selectlanguage{ngerman}±SEM. Student's t-test or one-way ANOVA with Tukey post hoc
analysis, *P\textless{}0.05; n=5-6 per group.

\textbf{Figure 5. Effects of low dose chronic treatments I2-IR LSL60101,
donepezil and co-administration on microgliosis and inflammatory markers
in 5XFAD mice. (a)} Representative images of GFAP immunostaining and
quantification \textbf{(b-d)} in DG, CA1, CA3 areas of the hippocampus
of the 5XFAd mice. Representative gene expression in the hippocampus of
the 5XFAD mice for \textbf{(e)} \selectlanguage{greek}\emph{Il-1β, Il-6, Ccl12,}\selectlanguage{english}
\textbf{(f)}for \emph{Trem2} . Gene expression levels were determined by
real-time PCR. Bars represent mean\selectlanguage{ngerman}±SEM. Student's t-test or one-way
ANOVA with Tukey post hoc analysis, *P\textless{}0.05; n=5-6 per group.

\textbf{Figure 6. Effects of low dose chronic treatments I2-IR LSL60101,
donepezil and co-administration on astrogliosis and synaptic plasticity
in 5XFAD mice. (a)} Representative images of GFAP immunostaining and
quantification \textbf{(b-d)} in DG, CA1, CA3 areas of the hippocampus
of the 5XFAd mice. Representative western blot and
quantifications\textbf{(f-h)} for PSD95 and SYN in the hippocampus of
5XFAD mice. Values in bar graphs are adjusted to 100\% for protein
levels of the control WT control or the control 5XFAD. Bars represent
mean±SEM. Student's t-test or one-way ANOVA with Tukey post hoc
analysis, *P\textless{}0.05; n=5-6 per group.

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