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Antioxidants 2023, 12, 111. https://doi.org/10.3390/antiox12010111 www.mdpi.com/journal/antioxidants
Article
Intranasal Administration of KYCCSRK Peptide Rescues Brain
Insulin Signaling Activation and Reduces Alzheimer’s
Disease-Like Neuropathology in a Mouse Model for
Down Syndrome
Antonella Tramutola
1
, Simona Lanzillotta
1
, Giuseppe Aceto
2,3
, Sara Pagnotta
1
, Gabriele Ruffolo
4,5
,
Pierangelo Cifelli
6
, Federico Marini
7
, Cristian Ripoli
2,3
, Eleonora Palma
4,5
, Claudio Grassi
2,3
,
Fabio Di Domenico
1
, Marzia Perluigi
1
and Eugenio Barone
1,
*
1
Department of Biochemical Sciences “A. Rossi-Fanelli”, Sapienza University of Rome, Piazzale A. Moro 5,
00185 Roma, Italy
2
Department of Neuroscience, Università Cattolica del Sacro Cuore, 00168 Roma, Italy
3
Fondazione Policlinico Universitario A. Gemelli, Istituto di Ricovero e Cura a Carattere Scientifico,
00168 Roma, Italy
4
Department of Physiology and Pharmacology, Istituto Pasteur-Fondazione Cenci Bolognetti,
University of Rome Sapienza, 00185 Rome, Italy
5
IRCCS San Raffaele Roma, 00163 Rome, Italy
6
Department of Applied Clinical and Biotechnological Sciences, University of L’Aquila, 67100 L’Aquila, Italy
7
Department of Chemistry, Sapienza University of Rome, Piazzale A. Moro 5, 00185 Roma, Italy
* Correspondence: eugenio.barone@uniroma1.it
Abstract: Down syndrome (DS) is the most frequent genetic cause of intellectual disability and is
strongly associated with Alzheimer’s disease (AD). Brain insulin resistance greatly contributes to
AD development in the general population and previous studies from our group showed an early
accumulation of insulin resistance markers in DS brain, already in childhood, and even before AD
onset. Here we tested the effects promoted in Ts2Cje mice by the intranasal administration of the
KYCCSRK peptide known to foster insulin signaling activation by directly interacting and
activating the insulin receptor (IR) and the AKT protein. Therefore, the KYCCSRK peptide might
represent a promising molecule to overcome insulin resistance. Our results show that KYCCSRK
rescued insulin signaling activation, increased mitochondrial complexes levels (OXPHOS) and
reduced oxidative stress levels in the brain of Ts2Cje mice. Moreover, we uncovered novel
characteristics of the KYCCSRK peptide, including its efficacy in reducing DYRK1A (triplicated in
DS) and BACE1 protein levels, which resulted in reduced AD-like neuropathology in Ts2Cje mice.
Finally, the peptide elicited neuroprotective effects by ameliorating synaptic plasticity mechanisms
that are altered in DS due to the imbalance between inhibitory vs. excitatory currents. Overall, our
results represent a step forward in searching for new molecules useful to reduce intellectual
disability and counteract AD development in DS.
Keywords: Alzheimer’s disease; brain insulin resistance; Down syndrome; DYRK1A; intellectual
disability
Citation: Tramutola, A.; Lanzillotta,
S.; Aceto, G.; Pagnotta, S.; Ruffolo,
G.; Cifelli, P.; Marini, F.; Ripoli, C.;
Palma, E.; Grassi, C.; et al. Intranasal
Administration of KYCCSRK
Peptide Rescues Brain Insulin
Signaling Activation and Reduces
Alzheimer’s Disease-Like
Neuropathology in a Mouse Model
for Down Syndrome. Antioxidants
2023, 12, 111. https://doi.org/10.3390/
antiox12010111
Academic Editor: Stanley Omaye
Received: 19 December 2022
Revised: 29 December 2022
Accepted: 30 December 2022
Published: 2 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/license
s/by/4.0/).
Antioxidants 2023, 12, 111 2 of 27
1. Introduction
Down syndrome (DS) is the most frequent genetic cause of intellectual disability and
is strongly associated with Alzheimer’s disease (AD) [1]. It is a multifaceted disorder with
over 80 clinically defined phenotypes including those affecting the central nervous
system, heart, gastrointestinal tract, skeleton, and immune system [2]. Phenotypes
associated with trisomy 21 vary in both incidence and severity, leading to a vast array of
phenotypic combinations [3]. Prevalence of overweight can reach 70% in subjects with DS
leading to a higher incidence of type 2 diabetes mellitus (T2DM) and/or obesity [4].
Metabolic disorders are characterized by alterations in cell metabolism, e.g.,
mitochondrial defects, increased oxidative stress levels, impaired glucose, and lipid
metabolism, finally resulting in reduced energy production and cellular dysfunctions that
account for a higher incidence of diabetes [5,6] and/or obesity [7].
An intriguing epidemiological correlation between obesity, glucose dysmetabolism
and diabetes, and various complex brain diseases, including AD, exists. Diabetic subjects
develop insulin resistance, which leads to an increased risk of developing AD in later life
[8–10]. In a perfect storm, neuroinflammation, oxidative stress, and mitochondrial
dysfunction would aggravate brain insulin resistance and amyloid-beta (Aβ)
accumulation in brain lesions [8,10–12].
Remarkably, recent studies highlight that brain insulin resistance can develop even
independently from peripheral alterations both during aging and in AD, though the
molecular mechanisms are similar. Decreased insulin concentrations and insulin receptor
binding were reported in the cortex of elderly individuals without dementia [10,13] and
in AD patients without T2DM [14–18]. In two independent cohorts of post-mortem brain
samples isolated from individuals with AD or mild cognitive impairment (MCI),
substantial abnormalities were described in the basal activation of insulin signaling [14].
These abnormalities were negatively correlated with global cognition and memory scores
regardless of Aβ and Tau levels [16], suggesting that brain insulin resistance contributes
to cognitive impairment independently from AD neuropathology [15]. In support of these
observations, the evaluation of neuronal-derived extracellular vesicles (nEVs) cargo
demonstrated that increased markers of brain insulin resistance in nEVs predict the
development of AD in elderly individuals [19].
DS recapitulates several risk factors associated with brain insulin resistance
development, i.e., increased Aβ production and deposition due to triplication of the
amyloid precursor protein gene APP mapping to human chromosome 21 (HSA21), and
increased rates of obesity, glucose intolerance, and T2DM. Intriguingly, our group
showed the accumulation of insulin resistance markers in the brain in DS individuals
already in childhood, regardless of metabolic disorders, and even before AD development
[4,20–23]. Furthermore, brain insulin resistance manifests along with an impairment of
cell energy metabolism before frank AD pathological hallmarks accumulation both in
humans and DS mouse models [22,23], suggesting that a close link exists among these
alterations that contribute to brain dysfunctions and likely favor the development of AD
in DS.
Complex disorders pose tremendous challenges to research and healthcare. The
increasing number of patients with co- and multi-morbidities causes an urgent need to
improve care but also to develop new strategies to assess the progression of diseases, and
define socio-economic and lifestyle contributing factors. As brain insulin resistance has
been identified as a risk factor for AD, the concept has developed that drugs used to treat
peripheral insulin resistance (i.e., in T2DM or obesity) may have neuroprotective
properties [24,25]. Among the strategies to ameliorate the activation of insulin signaling
in the brain, intranasal administration of antidiabetic drugs is under evaluation in the field
of AD [26–29]. Intranasal administration represents an effective strategy that allows drugs
to bypass the blood-brain barrier (BBB) and directly reach the brain, thereby avoiding side
effects caused by systemic administration [30]. To note, intranasal insulin administration
promoted neuroprotective effects resulting in improved cognitive functions in MCI and
Antioxidants 2023, 12, 111 3 of 27
AD subjects [31–35]. Moreover, a recent pilot study showed that a single dose of intranasal
insulin was safe in DS individuals and promoted a trend toward improved performance
on memory retention [36], although further investigations are required due to the very
small sample size of the cohort of patients.
Successful intranasal delivery of biologics such as peptides, proteins, monoclonal
antibodies, oligonucleotides, and gene and cell therapies via the nose-to-brain route are
of increasing interest due to their high potency and selectivity [37,38]. Peptides
biodegrade into non-toxic metabolites, possess a minimal potential for drug-drug
interactions, are less likely to cause an immunogenic reaction when compared to larger
proteins, and have lower production costs [37–39]. These favorable properties have
resulted in peptides having a good probability of securing regulatory approval when
compared to low molecular weight drugs [38]. Moreover, their small size enables peptides
to penetrate the cell membrane to target intracellular molecules [39].
In searching for new potential neuroprotective molecules for DS, we focused on the
effects promoted by the KYCCSRK peptide. Previous works described an unprecedented
approach for stimulating insulin signaling activation and glucose uptake by means of the
KYCCSRK peptide, corresponding to the C-terminal 7 residues (K291YCCRSK) of the
human biliverdin reductase-A (BVR-A) protein [27,40,41], this latter being a key player in
the regulation of insulin signaling [42,43]. The KYCCSRK peptide was shown to promote
both the activation of the insulin receptor (IR) kinase activity and the activation of ERK1/2
and AKT downstream from IR [40,41] on its own in HEK cells, thus representing a
promising approach to counteract insulin resistance. Intriguingly, the KYCCSRK acts
predominantly at the intracellular level by favoring conformational changes of the IR
kinase domain in the β subunits and/or by interacting with ERK1/2 and AKT, thus
triggering their activation in the absence of insulin [40,41]. However, none of the above-
mentioned studies addressed the effects of the KYCCSRK peptide in vivo, neither in the
brain.
In the current work, we tested the hypothesis that the intranasal administration of
KYCCSRK peptide rescues brain insulin signaling activation and promotes
neuroprotective effects in the brain of Ts2Cje mice (a model for DS).
2. Materials and Methods
2.1. Mouse Colony
Ts2Cje (Rb(12.Ts171665Dn)2Cje) mice are a well-established murine model of DS
characterized by a triple copy of a Robertsonian fusion chromosome carrying the distal
end of Chr16 and Chr12. Parental generations were purchased from Jackson Laboratories
(Bar Harbour, ME, USA). The mouse colony was raised by a crossbreed of Ts2Cje trisomic
females with euploid (B6EiC3SnF1/J) F1 hybrid males (Eu). The parental generations were
purchased from Jackson Laboratories (Bar Harbour, ME, USA). These breeding pairs
produce litters containing both trisomic (Ts2Cje) and euploid (Eu) offspring. Pups were
genotyped to determine trisomy by standard PCR, using Reinoldth’s method [44]. Mice
were housed in clear Plexiglas cages (20 × 22 × 20 cm) under standard laboratory
conditions with a temperature of 22 ± 2 °C and 70% humidity, a 12-h light/dark cycle, and
free access to food and water.
2.2. KYCCSRK Treatment
Nine-month old male Ts2Cje and Eu mice were used in this study based on previous
results showing increased accumulation of brain insulin resistance and AD
neuropathological hallmarks at this age [22]. Euploid (Eu) and Ts2Cje (Ts-V) mice were
treated with vehicle (saline) (5μL/nostril), and an additional group of Ts2Cje (Ts-P) mice
received an intranasal administration of 0.5 mM N-myristoylated KYCCSRK peptide
(hereafter KYCCSRK) [PO#SP161119 Myr-KYCCSRK, (Biomatik, Delaware, USA] for two
weeks (n = 4/group). The choice to use a myristoylated peptide relies on the concept that
Antioxidants 2023, 12, 111 4 of 27
myristoylated peptides cross the plasma membrane and can modulate intracellular
processes, i.e., insulin signaling, as previously reported [40,41]. Based on the results
collected in a pilot dose-response treatment (0, 0.12, 0.25, 0.5, 1, and 2 mM) in Eu mice (n
= 3/group), we selected the dose of 0.5 mM, which was effective in stimulating the insulin
signaling in the frontal cortex, as demonstrated by the significantly increased activation
of the AKT-AS160-GLUT4 pathway (see Supplementary Figure S1).
2.3. Primary Neurons
For primary neuronal culture, Ts2cje and euploid pups at 0–1 post-natal day were
used. Regarding the collection of Ts2Cje primary neurons, because the breeding pairs
produce litters containing both trisomic (Ts2Cje) and euploid (Eu) offsprings, Ts2Cje pups
were selected by tail-genotyping. After the sacrifice, the cortex was dissected and
processed (Ts2Cje and Eu separately) following a specific procedure to obtain neuronal
cells. In detail, cortical tissues were mechanically dissociated in cold phosphate-buffered
saline (PBS) supplemented with Ca2+ and Mg2+ (Sigma-Aldrich, St. Louis, MO, USA) and
centrifuged at 1100 × rpm for 3 min at room temperature. The supernatant was removed
and a solution of PBS w/o Ca2+ and Mg2+ [Sigma-Aldrich, St. Louis, MO, USA] and 0.25%
Trypsin (GIBCO, Thermo Fisher Scientific, Waltham, MA USA) was added for chemical
tissue dissociation in a shaking water bath at 37 °C for 10 min. To inactivate the trypsin,
10% fetal bovine serum (FBS) (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) was
added to the dissociated tissues and centrifuged at 1100 x rpm for 3 min at room
temperature. The cell-pellet was resuspended in Minimum Essential Medium (MEM)
(GIBCO, Thermo Fisher Scientific, Waltham, MA USA) supplemented with 1% FBS, 1% L-
glutamine (200 mM, Sigma-Aldrich, St. Louis, MO, USA), 1% glucose (25 mM, Sigma-
Aldrich, St. Louis, MO, USA) and 1% Gentamicin (0,1 mg/mL, Sigma-Aldrich, St. Louis,
MO, USA) and centrifuged at 1100 × rpm for 10 min at room temperature. The obtained
pellet was resuspended in a second MEM supplemented with 5% FBS, 5% human serum,
1% L-glutamine (200 mM), 1% glucose (25 mM), and 1% gentamycin (0.1 mg/mL), and the
cells were plated at a density of 150,000 cells/well in a multi-well previously coated with
poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA). After 24 h the medium was replaced
with Neurobasal Medium (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA)
supplemented with 2% B-27 serum free (GIBCO, Thermo Fisher Scientific, Waltham, MA,
USA), 1% L-glutamine (200 mM, Sigma-Aldrich, St. Louis, MO, USA), and 1% gentamicin
(0.1 g/mL, Sigma-Aldrich, St. Louis, MO, USA) to start neuronal differentiation. After 4
days, the medium was replaced with Neurobasal Medium supplemented with 2% B-27
(GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) and 1% gentamicin (0,1 mg/mL,
Sigma-Aldrich, St. Louis, MO, USA) to let the neurons grow up. After 10 days from the
cell plating, the neurons were treated with the KYCCSRK peptide at different doses (1, 5,
and 20 μM) as previously described [27,40] (see Supplementary Figure S2). After 24 h cells
were collected and stored at −80 °C for the Western blot analysis described below.
2.4. Samples Preparation
Total protein extracts from either cortical primary neurons or Ts2Cje and Eu cortical
samples were prepared in radioimmunoprecipitation assay (RIPA) buffer (pH 7.4)
containing 50 mM Tris-HCl (pH 7.4, Sigma-Aldrich, St. Louis, MO, USA), 150 mM NaCl
(Sigma-Aldrich, St. Louis, MO, USA), 1% NP-40 (Sigma-Aldrich, St. Louis, MO, USA),
0.25% sodium deoxycholate (Sigma-Aldrich, St. Louis, MO, USA), 1 mM EDTA (Sigma-
Aldrich, St. Louis, MO, USA), 0,1% sodium dodecyl sulfate (SDS, Sigma-Aldrich, St. Louis,
MO, USA) and supplemented with phosphatase and protease inhibitors (1:100, Sigma-
Aldrich, St. Louis, MO, USA). Then, samples were homogenized by 20 strokes of a
Wheaton tissue homogenizer, sonicated, and centrifuged at 14.000 x rpm for 30 min at 4
◦C to remove debris. The supernatant was collected, and the total protein concentration
was determined by the BCA method according to manufacturer’s instructions (Thermo
Fisher Scientific, Waltham, MA, USA).
Antioxidants 2023, 12, 111 5 of 27
Antioxidants 2023, 12, 111 6 of 27
2.5. Western Blot
Fifteen μg of proteins were resolved via SDS-PAGE using Criterion™ TGX Stain-
Free™ precast gel (Bio-Rad Laboratories, Hercules, CA, USA) in a Criterion large format
electrophoresis cell (Bio-Rad Laboratories, Hercules, CA, USA) in Tris/Glycine/SDS (TGS)
Running Buffer (Bio-Rad Laboratories, Hercules, CA, USA). Immediately after electro-
phoresis, the gel was placed on a Chemi/UV/Stain-Free tray and then placed into a Chem-
iDoc MP imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and UV-activated
based on the appropriate settings with Image Lab Software (Bio-Rad Laboratories, Her-
cules, CA, USA) to collect total protein load image. Following electrophoresis and gel im-
aging, the proteins were transferred onto a nitrocellulose membrane by Trans-Blot Turbo
Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). To prove the transfer, the
blot was imaged by the ChemiDoc MP imaging system using the Stain-Free Blot settings.
The nitrocellulose membrane was blocked using 3% BSA (SERVA Electrophoresis GmbH,
Heidelberg, Germany) in 1X Tris Buffer Saline (TBS) containing 0.01% Tween20 (Sigma-
Aldrich, St. Louis, MO, USA) and incubated overnight at 4 °C with the primary antibodies
listed in Table 1. The day after, all membranes were washed with 1X TBS containing 0.01%
Tween20 (Sigma-Aldrich, St. Louis, MO, USA) and incubated at room temperature for 1
h with the respective secondary antibody conjugated with horseradish peroxidase: anti-
rabbit (1:10.000; Bio-Rad Laboratories, Hercules, CA, USA), anti-mouse (1:10.000; Bio-Rad
Laboratories, Hercules, CA, USA). Membranes were developed with Clarity enhanced
chemiluminescence (ECL) substrate (Bio-Rad Laboratories, Hercules, CA, USA) and then
acquired with Chemi-Doc MP (Bio-Rad, Hercules, CA, USA) and analyzed using Image
Lab 6.1 software (Bio-Rad, Hercules, CA, USA) that allows the normalization of a specific
protein signal by the mean of total proteins load. Total protein staining measures the ag-
gregate protein signal (sum) in each lane and eliminates the error that can be introduced
by a single internal control protein. Total protein staining is a reliable and widely appli-
cable strategy for quantitative immunoblotting. It directly monitors and compares the ag-
gregate amount of sample protein in each lane, rather than using an internal reference
protein as a surrogate marker of sample concentration. This direct, straightforward ap-
proach to protein quantification may increase the accuracy of normalization. Total load
can be detected taking advantage of the Stain free technology (Bio-Rad, Hercules, CA,
USA). Stain-free imaging technology utilizes a proprietary trihalo compound to enhance
natural protein fluorescence by covalently binding to tryptophan residues with a brief UV
activation. Images of the gel or membrane after transfer can easily be captured multiple
times without staining and de-staining steps.
Table 1. Antibodies used in the current work.
Name Code Company Dilution
3-NT SAB5200009 Sigma-Aldrich 1:1000
4-HNE NB10063093 Novus 1:2000
4EBP1 (P1) sc-9977 SANTA CRUZ 1:1000
Akt AM-1011 ECM 1:1000
ADAM10 Ab227172 Abcam 1:1500
APP A8717 Sigma-Aldrich 1:10000
APP-C99 MABN380 Sigma-Aldrich 1:1000
ATG5 sc-133158 SANTA CRUZ 1:1000
BACE1 sc-33711 SANTA CRUZ 1:1000
Beclin 3738 Cell signaling 1:1000
BVR-A Ab90491 Abcam 1:5000
CamKIIα Sc-32288 Santa Cruz 1:1000
DYRK1A 8765S Cell signaling 1:1000
GluA1 2263 Millipore 1:1000
Antioxidants 2023, 12, 111 7 of 27
GLUT 4 (IF-8) sc 53566 SANTA CRUZ 1:1000
IR 3020S Cell signaling 1:1000
LC3B NB-1002220 Novus 1:1000
NdufB6 PA5-60579 ThermoFisher 1:2000
OXPHOS Ab110411 ABCAM 1:2000
p4EBP1 (T36) sc-18080-R SANTA CRUZ 1:500
pAkt (S473) 44621 Invitrogen 1:1000
pAS160 (T642) GTX 55118 Gene Tex 1:1000
pCamKIIα (T286) 12716 Cell signaling 1:1000
pGluA1 (S831) 4-833 Millipore 1:1000
pGluA1 (S845) 8084 Cell signaling 1:1000
pIR (Y1146/Y1150/Y1151) GTX25681 Gene Tex 1:1000
pIRS1 (S307) 2381 Cell signaling 1:1000
pIRS1 (S636) GTX 32400 Gene Tex 1:1000
pIRS1 (Y612) GTX24868 Gene Tex 1:1000
pmTOR (S2448) 5536S Cell signaling 1:1000
pPTEN (S380/T382/383) sc-101789 SANTA CRUZ 1:500
Protein Carbonyl S7150 Sigma-Aldrich 1:5000
PSD95 D27E11 Cell signaling 1:1000
pTAU (AT8) MN1020 Invitrogen 1:2000
pTAU (S404) Ab92676 ABCAM 1:1000
PTEN (A2B1) sc-7974 SANTA CRUZ 1:1000
SQSTM1 GTX100685 Genetex 1:1000
Synaptophysin 8049 Abcam 1:1000
Tau orb-46243 Biorbyt 1:1000
VDAC PA1-954A Invitrogen 1:1000
2.6. Slot Blot
For the analysis of total protein carbonyls (PC) levels, 5 μL of cortical total protein
extract samples were derivatized with 5 μL of 10 mM 2,4-dinitrophenylhydrazine (DNPH,
OxyBlot™ Protein Oxidation Detection Kit, Merck-Millipore, Darmstadt, Germany) in the
presence of 5 μL of 10% sodium dodecyl sulfate (SDS) for 20 min at room temperature.
The samples were then neutralized with 7.5 μL of neutralization solution (2M Tris in 30%
glycerol) and loaded onto a nitrocellulose membrane as described below.
For total (i) protein-bound 4-hydroxy-2-nonenals (HNE) and (ii) 3-nitrotyrosine (3-
NT) levels: 5 μL of cortical total protein extract samples, 5 μL of 12% SDS, and 5 μL of
modified Laemmli buffer containing 0.125 M Tris base (pH 6.8), 4% (v/v) SDS, and 20%
(v/v) glycerol were incubated for 20 min at room temperature and then loaded onto nitro-
cellulose membrane as described below.
Proteins (250 ng) were loaded in each well on a nitrocellulose membrane under vac-
uum using a slot blot apparatus. Membranes were blocked for 1 h with a solution of 3%
(w/v) BSA in PBS containing 0.01% (w/v) sodium azide and 0.2% (v/v) Tween 20 and incu-
bated respectively with primary antibodies anti-HNE and anti-3NT (details are listed in
Table 1) for 2 h at RT. Membranes were washed in TTBS following primary antibody in-
cubation three times at intervals of 5 min each and then incubated with anti-rabbit or
mouse IgG alkaline phosphatase secondary antibodies (Sigma-Aldrich, St Louis, MO,
USA) for 1 h at room temperature. Then, membranes were washed three times in TBS
solution containing 0.01% Tween 20 for 5 min each and developed with Sigma fast tablets
(5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate [BCIP/NBT sub-
strate]). Membranes were dried and the images were acquired using the ChemiDoc XP
image system and analyzed using Image Lab software (Bio-Rad Laboratories, Hercules,
CA, USA). No non-specific binding of the antibody to the membrane was observed.
Antioxidants 2023, 12, 111 8 of 27
2.7. Real-Time PCR
RNA was obtained from cortical tissue of Ts2Cje and Eu mice treated with vehicle or
th e KYCCS RK pept ide. Ti ssues w ere lys ed with an appropri ate vol ume of Q IAzol R eagent
(QIAzol Lysis Reagent, Qiagen, Hilden, Germany). Subsequently, chloroform was added
(1:5, Sigma-Aldrich, St. Louis, MO, USA), and samples were kept for 3 min at RT before
being centrifuged at 12,000 × rpm and 4 °C for 15 min. Following that, isopropanol (100%,
Sigma-Aldrich, St. Louis, MO, USA) was added to each sample at RT for 10 min to sepa-
rate RNA. Then, samples were centrifuged at 12,000 rpm and 4 °C for 15 min. The super-
natant was discarded, and pellets were washed with 75% ethanol (Sigma-Aldrich, St.
Louis, MO, USA) followed by further centrifugation at 7500 × rpm and 4 ◦C for 5 min. The
pellets were resuspended in RNAse-free H2O (Sigma-Aldrich, St. Louis, MO, USA). The
RNA was quantified using the Biospec Nano reverse transcribed using the cDNA High-
Capacity kit (Applied Biosystems, Foster City, CA, USA), including reverse transcriptase,
random primers, and buffer according to the manufacturer’s instructions. The cDNA was
produced through a series of heating and annealing cycles in the MultiGene OPTIMAX
96-well Thermocycler (LabNet International, Edison, NJ, USA). Real-time PCR was car-
ried out using the SensiFAST™ SYBR® and No-ROX Kit (Bioline, London, UK) in a CFX
Connect Real-Time PCR machine (Bio-Rad Laboratories, Hercules, CA, USA). Primers
used for the RT-PCR are DYRK1A FW: 5′-TGGGGCAGAGGATATACCAGT-3′ and RV:
5′- GTCGATAGCAAGGTCATAAGGCA-3′ and BACE1 FW 5′-GGAT-
TATGGTGGCCTGAGCA-3′ RV 5′-CACGAGAGGAGACGACGATG-3′ (BACE1:
NM_011792.7; DYRK!A: NM_007890.2).
2.8. Membrane Preparation, Microtransplantation, and Xenopus Oocytes Recordings
Xenopus oocytes were obtained from adult female frogs. The procedure for the oo-
cytes’ preparation was described elsewhere [45]. Then, the cytoplasmatic injection was
performed with a pressure microinjector (PLI-100, Warner Instruments, Holliston, MA,
USA). Cell membranes were prepared from cortex of Eu, Ts-V, and Ts-P mice (n = 4/group)
that were immediately processed after their removal or stored at −80 °C. Then, the injected
oocytes were maintained in modified Barth’s solution at 16 °C until electrophysiological
recordings were performed. Xenopus frogs were purchased from Xenopus1 (USA). The
use of Xenopus laevis frogs, and the surgical procedures for oocytes preparation and use
conformed to the Italian Ministry of Health guidelines and were approved by the same
institution (authorization no 427/2020-PR).
2.9. Voltage-Clamp Recordings
Experiments with microtransplanted oocytes were carried out 24–48 h after cytoplas-
mic injection and GABA and AMPA-evoked currents (IGABA and IAMPA) were recorded us-
ing the technique of two-electrode voltage clamp. The microelectrodes were filled with 3
M KCl [46] and oocytes placed in a recording chamber (0.1 mL volume) continuously per-
fused with oocyte Ringer solution (OR: 82.5 mM NaCl; 2.5 mM KCl; 2.5 mM CaCl2; 1 mM
MgCl2; 5 mM Hepes, adjusted to pH 7.4 with NaOH) at room temperature (20–22 °C). The
timing of neurotransmitter application was controlled by a gravity driven multi-valve
perfusion system (9–10 mL/min) which was controlled through a computer interface (Bi-
ologique RSC- 200; Claix, France) to ensure the exact duration of each application. With
our setup, 0.5 to 1 s was enough to completely replace the entire volume of the applied
solution in the recording chamber.
In these experiments, 500 μM GABA was applied for 5 s followed by a washout of 4–
5 min. To record AMPA currents, we used a preincubation of 20 s with cyclothiazide (CTZ,
20 μM to avoid AMPA’s receptor desensitization) before applying 20 μM AMPA plus CTZ
for 10 s [47]. The stability of the IGABA and IAMPA was ascertained by performing two con-
secutive GABA and AMPA applications, separated by a 4–5 min washout. The cells that
had a <5% variation of current amplitude were considered in the statistical analysis.
Antioxidants 2023, 12, 111 9 of 27
2.10. Statistical Analyses
Statistical analyses were performed by using one-way ANOVA analysis with Dun-
nett’s or Kruskal–Wallis multiple comparison tests for the evaluation of differences be-
tween more than two groups. All statistical tests were two-tailed and the level of signifi-
cance was set at 0.05. Data are expressed as mean ± SEM per group. Correlation analyses
were calculated by Pearson’s rank correlations and displayed as a correlation matrix. Fur-
thermore, principal component analysis (PCA) was used. PCA is a statistical method for
data compression and visualization. Multivariate data are projected along orthogonal di-
rections (principal components) along which they possess the maximum variance. Simi-
larity and dissimilarities among samples are inspected by plotting the new coordinates of
the data along the principal components (scores), while interpretation of the observed dif-
ferences is achieved by investigating the variable contributions to the new directions
(loadings). More specifically, PCA analysis was conducted on the data matrix after col-
umn autoscaling by means of in-house written routines running under Matlab (R2015b;
The Mathworks, Natick, MA, USA) environment. All the other statistical analyses were
performed using Graph Pad Prism 9.0 software (GraphPad, La Jolla, CA, USA).
3. Results
3.1. Intranasal KYCCSRK Administration Rescues Insulin Signaling Activation in the Frontal
Cortex of Ts2Cje Mice
To evaluate the effects of the intranasal KYCCSRK peptide administration on the
brain insulin signaling pathway in Ts2Cje mice, we started by measuring protein levels
and activation of IR and IRS1, the latter being the main target of IR kinase activity [10]. IR
protein levels were significantly increased in Ts-V compared to Eu mice (+64%, p= 0.021),
while no changes were induced by KYCCSRK peptide treatment (Ts-P) (Figure 1B). Acti-
vation of IR (evaluated as pIRY1146/1150/1151/IR ratio) was significantly reduced in Ts-V
with respect to Eu mice (−54% vs. Eu, p = 0.023), while the KYCCSRK administration res-
cued IR activation in Ts-P mice (+64 %, p = 0.014) (Figure 1C). Regarding IRS1 total protein
levels, no significant changes were observed among groups (Figure 1D). No changes for
IRS1 activation (evaluated as pIRS1Y612/IRS1 ratio) were observed (Figure 1E). IRS1 inhi-
bition was evaluated by the mean of S636 and S307 phosphorylation sites (reported as
pIRS1S636/IRS1 and pIRS1S307/IRS1 ratio, respectively), which are well known markers
of insulin resistance [48,49]. We found that S636 phosphorylation levels were not different
between Ts-V and Eu, while S307 phosphorylation levels were significantly increased in
Ts-V mice (+258% vs. Eu, p = 0.001) (Figure 1F,G). Rather, the intranasal KYCCSRK treat-
ment led to a consistent reduction of both S636 (−60% vs. Ts-V, p = 0.01) and S307 phos-
phorylation levels (−163% vs. Ts-V, p = 0.02) in Ts-P mice (Figure 1F,G). These data suggest
that brain insulin resistance develops in Ts2Cje mice brain while the KYCCSRK peptide
administration rescues IR/IRS1 activation.
Antioxidants 2023, 12, 111 10 of 27
Figure 1. Activation of insulin signaling following KYCCSRK intranasal administration in Ts2Cje
mice. IR, IRS1, AKT, BVR-A, and PTEN protein levels and phosphorylation were evaluated in the
frontal cortex of Eu and Ts2Cje (Ts-V) mice treated with vehicle (saline) and Ts2Cje (Ts-P) treated
with KYCCSRK peptide (0.5 mM) for two weeks (n = 4/group). (A) Representative Western blot and
total load images and densitometric evaluation of (B,C) IR protein levels and activation [evaluated
as pIRY1146/1150/1151/IR ratio], (D) IRS1 protein levels, (E) IRS1 activation evaluated as pIRS1Y612/IRS1
ratio], (F,G) IRS1 inhibition [evaluated as pIRS1S636/IRS1 and pIRS1S307/IRS1 ratio], (H,I) AKT protein
levels and activation (evaluated as pAKTS473/AKT ratio), (J) BVR-A protein levels, and (K,L) PTEN
protein levels and inhibition (evaluated as pPTENS380/T382/383/PTEN). All densitometric values were
normalized per total protein load and are given as a percentage of Eu set as 100%. Data are shown
as mean ± SEM. One-way ANOVA with Dunnett test: * p <0.05, ** p < 0.01.
To test whether the observed KYCCSRK-induced IR and IRS1 activation was associ-
ated with the stimulation of the entire signaling, we evaluated AKT levels and activation
since AKT is one of the main intracellular proteins activated in response to insulin [48,49].
Our results show no changes for AKT protein levels (Figure 1H), while increased AKT
phosphorylation (evaluated as pAktS473/Akt ratio), and thus activation, in Ts-P mice, was
observed (+319% vs. Ts-V, p = 0.05) (Figure 1I).
Finally, to better clarify the molecular mechanisms responsible for the observed ef-
fects mediated by the KYCCSRK administration in Ts2Cje mice, we focused on two key
proteins regulating insulin signaling activation: the BVR-A and the phosphatidylinositol-
3,4,5-trisphosphate 3-phosphatase (PTEN).
BVR-A is one of the main regulators of insulin signaling, that forms a regulatory loop
together with IR and IRS1 [27,50], while downstream from IR/IRS1 it favors the activation
of several proteins including ERK1/2, Akt, GSK-3β, PKCζ and mTOR [42,51–54]. As ex-
plained above, the KYCCSRK peptide corresponds to the C-terminal 7 residues of human
BVR-A, so we were interested in understanding whether the peptide might affect mouse
BVRA protein levels. We found no changes for BVRA either in Ts-V mice vs. Eu or in Ts-
P mice (Figure 1J), suggesting that BVRA functions are preserved following the treatment.
PTEN is a phosphatase, that regulates the activation of the insulin signaling pathway
by reducing levels of PI3K-derived phosphatidylinositol (3,4,5)-trisphosphate (PIP3),
which promotes AKT activation [55,56]. Phosphorylation at the level of S380/T382/383, the
A
Eu
Ts-V
Ts-P
0
50
100
150
200
IR protein levels
(% of Eu)
*
Eu
Ts-V
Ts-P
0
50
100
150
pIR/IR ratio
(% of Eu)
**
Eu
Ts-V
Ts-P
0
50
100
150
pIRS1
S636
/IRS1 ratio
(% of Eu)
*
Eu
Ts-V
Ts-P
0
100
200
300
pIRS1
Y612
/IRS1 ratio
(% of Eu)
Eu
Ts-V
Ts-P
0
50
100
150
200
250
AKT protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
200
400
600
800
pAKT
S473
/AKT ratio
(% of Eu)
*
BC
EFK
HI
D
J
G
L
Eu
Ts-V
Ts-P
0
40
80
120
IRS1 protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
100
200
300
400
500
pIRS1
S307
/IRS1 ratio
(% of Eu)
** *
Eu
Ts-V
Ts-P
0
50
100
150
BVR-A protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
40
80
120
PTEN protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
40
80
120
pPTEN/PTEN ratio
(% of Eu)
Regulatory proteins
Antioxidants 2023, 12, 111 11 of 27
sites evaluated in the present study, is responsible for PTEN inhibition [56]. No changes
were observed for PTEN levels and activation (Figure 1K,L).
Together, these results highlight that KYCCSRK peptide administration rescues
brain insulin signaling activation both at levels of IR/IRS1 and AKT, without affecting
regulatory proteins, in agreement with previous observations in vitro [27,40] and in vivo
[41]. Rescuing brain insulin signaling activation might promote neuroprotective effects by
reducing AD neuropathological hallmarks in the brain, by improving synaptic plasticity
mechanisms, and by stimulating cell energy metabolism, which are all processes known
to be impaired in DS [4,57].
3.2. Intranasal KYCCSRK Administration Reduces the Accumulation of AD Neuropathological
Hallmarks in Ts2Cje Mice
AD neuropathological hallmarks, such as APP, APP cleavage products, and TAU
phosphorylation, accumulate quite early in the brain of human and mouse models of DS,
representing neurotoxic stimuli [1,22,23,58]. Indeed, the APP gene is triplicated in DS and
is a major risk factor for AD development in DS [57]. In addition, alterations of brain in-
sulin signaling were proposed to accelerate the development of AD pathology by promot-
ing either the aberrant cleavage of APP or TAU hyper-phosphorylation in the brain
[10,59]. Hence, to understand whether KYCCSRK peptide administration in Ts2Cje mice
could impact the accumulation of AD neuropathological hallmarks in the brain, APP full
length (APP), APP cleavage products (APP-C83 and APP-C99), and TAU levels and phos-
phorylation were evaluated.
Our results confirmed that APP (+60% vs. Eu, p = 0.0009) (Figure 2B) and its cleavage
products, i.e., APP-C99 (+111% vs. Eu, p = 0.0001) and APP-C83 (+42% vs. Eu, p = 0.02) are
elevated in the brain of Ts-V mice (Figure 2C,D). Following the treatment, a significant
reduction of both APP-C99 (−60% vs. Ts-V, p = 0.005) and APP-C83 (−92% vs. Ts-V, p =
0.0002) levels in Ts-P mice was observed (Figures 2C,D). To explain the mechanism
through which the KYCCSRK peptide administration reduces APP processing, we evalu-
ated protein levels of the enzymes α-secretase (ADAM-10) and β-secretase 1 (BACE1), that
are responsible for the production of APP-C83 and APP-C99, respectively [60]. We ob-
served a significant increase of the mature form (active) of ADAM-10 (mADAM-10) in Ts-
V mice (+84% vs. Eu, p = 0.05), regardless of the immature form (proADAM-10), whose
protein levels do not change among the groups (Figures 2E,F). No significant changes for
active ADAM-10 were detected after the KYCCSRK peptide administration (Figure 2F).
Conversely, a significant decrease of BACE1 protein levels in Ts-P (−43% vs. Ts-V, p =
0.013) was observed (Figure 2G).
Antioxidants 2023, 12, 111 12 of 27
Figure 2. Reduced AD neuropathological hallmarks following KYCCSRK intranasal administration
in Ts2Cje mice. APP, APP-CTFs, BACE1, TAU and DYRK1A protein levels and phosphorylation
were evaluated in the frontal cortex of Eu and Ts2Cje (Ts-V) mice treated with vehicle (saline) and
Ts2Cje (Ts-P) treated with KYCCSRK peptide (0.5 mM) for two weeks (n = 4/group). (A) Representa-
tive Western blot and total load images and densitometric evaluation of (B–D) APP, -C83 and -C99
fragments protein levels, (E,F) immature (proADAM-10) and mature (mADAM-10) forms of
ADAM-10 protein levels, (G) BACE1 protein levels, (H–J) TAU protein levels, AT8 and S404 phos-
phorylation, and (K) DYRK1A protein levels. All densitometric values were normalized per total
protein load and are given as a percentage of Eu set as 100%. In (L,M) BACE1 and DYRK1A mRNA
levels are shown. Data are shown as mean ± SEM. One-way ANOVA with Dunnett test: * p < 0.05,
** p < 0.01, *** p < 0.001.
We found no differences for TAU protein levels (Figure 2H), nor for TAU S202/T205
(AT8) phosphorylation (Figure 2I). Notably, a significant reduction of TAU S404 phos-
phorylation in Ts-P mice was observed (−40% vs. Ts-V, p = 0.04) (Figure 2J). To clarify this
aspect, we evaluated DYRK1A protein, a kinase involved in TAU phosphorylation [61,62]
whose gene is triplicated in DS [63,64]. Intriguingly, our data show that DYRK1A protein
levels were elevated in Ts-V mice (+65% vs. Eu, p = 0.024), while a significant reduction
was observed following the KYCCSRK administration (−61% vs. Ts-V, p = 0.025) (Figure
2K).
To better understand the process responsible for reduced BACE1 and DYRK1A pro-
tein levels following the treatment, their gene expression was evaluated. Reduced BACE1
mRNA levels in Ts-P (−23% vs. Ts-V, p = 0.01) were observed (Figure 2L). No differences
were found for DYRK1A (Figure 2M).
In addition, we evaluated changes of proteins normally regulating autophagy. Au-
tophagy is known to be impaired in the DS brain contributing to the accumulation of dam-
aged proteins which form toxic aggregates triggering AD development in DS [65]. No
significant changes were observed in Ts-P mice, possibly suggesting that KYCCSRK treat-
ment does not affect the autophagic process, and thus the observed decrease of either
APP-CTFs or pTAU levels might be mainly due to reduced BACE1 and DYRK1A protein
levels (Supplementary Figure S3).
Finally, because the robust effects mediated by the KYCCSRK administration on
APP/BACE1 and DYRK1A were quite intriguing and unexpected for us, we strengthened
Eu
Ts-V
Ts-P
0
50
100
150
200
proADAM-10 protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
50
100
150
200
250
mADAM-10 protein levels
(% of Eu)
*
Eu
Ts-V
Ts-P
0
50
100
150
200
250
APP-C99
(% of Eu)
*****
Eu
Ts-V
Ts-P
0
50
100
150
200
APP protein levels
(% of Eu)
***
B
Eu
Ts-V
Ts-P
0
40
80
120
BACE1 protein levels
(% of Eu)
*
CD
E
Eu
Ts-V
Ts-P
0
50
100
150
TAU protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
40
80
120
160
200
pTAU AT8
(% of Eu)
Eu
Ts-V
Ts-P
0
50
100
150
pTAU
S404
(% of Eu)
*
Eu
Ts-V
Ts-P
0
50
100
150
200
DYRK1A protein levels
(% of Eu)
*
*
FG
HIJ
K
L
Eu
Ts-V
Ts-P
0
40
80
120
160
APP-C83
(% of Eu)
***
*
A
Gene expression
M
Antioxidants 2023, 12, 111 13 of 27
these observations by performing in vitro analyses. APP, APP-CFTs, BACE1, and
DYRK1A levels were evaluated in Ts2Cje primary cortical neurons treated with different
doses of KYCCSRK. Results collected in neurons confirmed what we have observed in
Ts2Cje mice, by showing no changes for APP but reduced APP-CTFs, BACE1, and
DYRK1A protein levels (Supplementary Figure S2).
In summary, the intranasal administration of the KYCCSRK peptide promoted an
improvement in terms of AD pathology by reducing the amyloidogenic cleavage of APP
as well as TAU phosphorylation likely mediated by reducing the expression levels of
BACE1 and DYRK1A proteins.
3.3. Intranasal KYCCSRK Administration Reduces Oxidative Stress Levels in Ts2Cje Mice
In previous works from our group, we highlighted a harmful synergistic effect be-
tween brain insulin resistance and oxidative stress in DS [22,23]. Both brain insulin re-
sistance and increased oxidative stress levels in the brain were observed before the accu-
mulation of APP-C99 and TAU phosphorylation, suggesting these events perhaps accel-
erate AD development in DS [22,23,66]. For that reason, we hypothesized that the benefi-
cial effects of the KYCCSRK peptide treatment in Ts2Cje mice on brain insulin signaling
might be associated with a reduction of oxidative stress levels. We evaluated the levels of
three protein oxidation markers, named protein carbonyls (PC), 4-hydroxy-2-nonenal pro-
tein adducts (4-HNE), and protein-bound 3-nitrotyrosine (3-NT) in the frontal cortex of
Ts2Cje and Eu mice. Our results show increased PC (+20%, p = 0.032) (Figure 3A), 4-HNE
(+108%, p = 0.004) (Figure 3B) and 3-NT levels (+73%, p = 0.0152) (Figure 3C) in Ts-V mice
that were significantly reduced following the KYCCSRK treatment in Ts-P mice: PC
(−20%, p = 0.030), 4-HNE (−72%, p = 0.046) and 3-NT (−68%, p = 0.020) (Figure 3A–C).
Figure 3. Reduced oxidative stress markers levels following KYCCSRK intranasal administration in
Ts2Cje mice. (A) PC, (B) 4-HNE and (C) 3-NT levels were evaluated in the frontal cortex of Eu and
Ts2Cje (Ts-V) mice treated with vehicle (saline) and Ts2Cje (Ts-P) mice treated with KYCCSRK pep-
tide (0.5 mM) for two weeks (n = 4/group). Data were expressed as the percentage of Eu set as 100%.
Data are shown as mean ± SEM. One-way ANOVA with Dunnett test: * p <0.05, ** p < 0.01.
3.4. Intranasal KYCCSRK Administration Increases Mitochondrial Complexes Levels in
Ts2Cje Mice
Mitochondria are the powerhouse of cells responsible for the generation of ATP
(through oxidative phosphorylation) and the maintenance of redox homeostasis, as well
as for cell energy metabolism processes [67,68]. Genes involved in the oxidative phosphor-
ylation process are mostly downregulated in DS consistent with the development of sig-
nificant metabolic disturbances and increased oxidative stress levels in DS [69,70]. Fur-
thermore, we found that reduced levels of mitochondrial complexes proteins were signif-
icantly associated with higher brain insulin resistance markers levels and increased oxi-
dative stress levels both in human and mouse brains [22,23]. Hence, we hypothesized that
Antioxidants 2023, 12, 111 14 of 27
the beneficial effects mediated by the KYCCSRK peptide on the insulin signaling path-
way, AD neuropathological hallmarks, and oxidative stress levels in Ts2Cje mice, might
be associated with an improvement of mitochondrial machinery.
We evaluated changes of mitochondrial complexes (I–IV), and ATP synthase
(ATPase/CV) levels along with changes of voltage-dependent anion-selective channel
(VDAC), a protein that plays a key role in maintaining high rates of oxidative phosphor-
ylation [71]. We found that CI, CII, CIII, and ATPase levels showed a trend of reduction
in the frontal cortex of Ts-V mice compared to Eu, although they did not reach statistical
significance (Figure 4). Remarkably, KYCCSRK peptide administration promoted a sig-
nificant increase of CI (+115% vs. Ts-V, p = 0.05) (Figure 4C) and CIII levels (+130%, p =
0.05) (Figure 4F) along with a trend observed for both CII and ATP synthase (+50% and
+52%, respectively, vs. Ts-V) (Figures 4E–G). No changes for VDAC were observed.
Figure 4. Changes of mitochondrial proteins following KYCCSRK intranasal administration in
Ts2Cje mice. Mitochondrial OXPHOS complexes and VDAC protein levels were evaluated in the
frontal cortex of Eu and Ts2Cje (Ts-V) mice treated with vehicle (saline) and Ts2Cje (Ts-P) mice
treated with KYCCSRK peptide (0.5 mM) for two weeks (n = 4/group). (A) Representative Western
blot and total load images and densitometric evaluation of (B) VDAC protein levels, (C–G) mito-
chondrial complexes (I–IV) and ATPase related to VDAC. All densitometric values were normalized
per total protein load and are given as a percentage of Eu set as 100%. Data are shown as mean ±
SEM. One-way ANOVA with Dunnett test: * p < 0.05.
Together, these results support the hypothesis that improving brain insulin signaling
stimulates mitochondrial activation and cell energy metabolism in the Ts2Cje frontal cor-
tex.
3.5. Intranasal KYCCSRK Administration Improves Synaptic Plasticity Mechanisms in
Ts2Cje Mice
Brain insulin resistance impairs synaptic plasticity mechanisms, thus affecting learn-
ing and memory functions [10,72]. DS is characterized by a marked intellectual disability
and studies in DS mouse models show that an imbalance between inhibitory vs. excitatory
signals, likely due to an impairment of synaptic input integration [73], is responsible for
cognitive decline in DS. Moreover, we previously demonstrated that brain insulin re-
sistance is associated with alterations of mechanisms regulating synaptic plasticity both
in DS human and mouse brains [20,22,23]. Hence, to determine whether the observed im-
provement of brain insulin signaling also was associated with an amelioration of synaptic
plasticity mechanisms in Ts2Cje frontal cortex, changes of the GABA and AMPA currents
along with synaptic proteins i.e., synaptophysin, PSD95, CamKIIα, and GluA1R, were
evaluated.
A
Eu
Ts-V
Ts-P
0
50
100
150
VDAC protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
100
200
300
CI/VDAC ratio
(% of Eu)
*
Eu
Ts-V
Ts-P
0
50
100
150
CII/VDAC ratio
(% of Eu)
Eu
Ts-V
Ts-P
0
50
100
150
200
CIII/VDAC ratio
(% of Eu)
Eu
Ts-V
Ts-P
0
100
200
300
CIV/VDAC ratio
(% of Eu)
*
Eu
Ts-V
Ts-P
0
50
100
150
200
ATPase/VDAC ratio
(% of Eu)
DC
EGF
B
ATP a se
VDAC
kDa
Total Load
CI
CIV
CII
CIII
37
54
29
22
48
15
Eu Ts- V Ts - P
Antioxidants 2023, 12, 111 15 of 27
We used the microtransplantation of exogenous cell cortical membranes in Xenopus
oocytes that permits us to record evoked-currents from native receptors maintaining their
original characteristics [45–47]. Using this simple but powerful approach, differences of
the balance excitation/inhibition represented as AMPA/GABA currents percentage ratio
were evaluated. A higher AMPA/GABA ratio in the Ts-V (106 ± 10.32%, oocytes n = 20)
compared to the Eu mice (46.53 ± 5.84%, oocytes n = 12; p < 0.05) was observed. Notably,
Ts-P mice showed a lower AMPA/GABA ratio compared to Ts-V (43.74 ± 4.1%, oocytes n
= 13, p < 0.05), and were not statistically different from the Eu mice (Figure 5A), suggesting
that KYCCSRK intranasal administration restores the physiological ratio between IAMPA
e IGABA in Ts-P mice.
Figure 5. Improved synaptic plasticity mechanisms following KYCCSRK intranasal administration
in Ts2Cje mice. AMPA and GABA currents in oocytes transplanted with cortical membranes along
with proteins regulating synaptic plasticity mechanisms were evaluated in Eu and Ts2Cje (Ts-V)
mice treated with vehicle (saline) and Ts2Cje (Ts-P) mice treated with KYCCSRK peptide (0.5 mM)
for two weeks (n = 4/group). (A) Bars represent the mean ± SEM of the ratio between IAMPA and IGABA
(reported as percentage) in Eu (oocytes n = 12), Ts-V (oocytes n = 20) and Ts-P (oocytes n = 13) mice,
as shown. GABA 500 μM was applied for 4 s, while AMPA 20 μM plus CTZ were applied for 10 s.
Holding potential was −60 mV and * shows statistical significance (Kruskal–Wallis One Way Anal-
ysis of Variance on Ranks with Dunn’s test, p < 0.05). The mean of IAMPA was 7.2 ± 0.9 nA, 34.9 ± 3.9
nA, 11.4 ± 2.4 nA in Eu, Ts-V and Ts-P, respectively. The mean of IGABA was 16.7 ± 2.3 nA, 35.3 ± 3.7
nA, 25.9 ± 4.8 in Eu, Ts-V and Ts-P, respectively. Note the similar values in Eu and Ts-P. (B) Repre-
sentative current traces recorded in oocytes of the experiments shown in (A) as indicated; white
bars, GABA evoked-currents; black bars, AMPA evoked-currents. (C) Representative Western blot
and total load images and densitometric evaluation of (D) GluA1 total protein levels, (E,F) GluA1
phosphorylation at S831 and S845 (reported as pGluA1S831/GluA1 and pGluA1S845/GluA1 ratio);
(G,H) CamKIIα protein levels and activation (evaluated as pCamKIIαT286/CamKIIα ratio); (I) syn-
aptophysin and (J) PSD95 protein levels. All densitometric values were normalized per total protein
load and are given as a percentage of Eu set as 100%. Data are shown as mean ± SEM. One-way
ANOVA with Dunnett test: * p < 0.05 and ** p < 0.01.
To explain changes of AMPA currents we looked at the AMPA receptor GluA1 sub-
unit phosphorylation. Indeed, phosphorylation of the GluA1 subunit regulates activity-
Eu
Ts-V
Ts-P
0
50
100
150
200
250
pGluA1
S831
/GluA1 ratio
(% of Eu)
*
*
Eu
Ts-V
Ts-P
0
40
80
120
GluA1 protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
40
80
120
Synaptophysin protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
40
80
120
PSD95 protein levels
(% of Eu)
Eu
Ts-V
Ts-P
0
100
200
300
400
500
pCamKII
α
T286
/CamKII
α
ratio
(% of Eu)
** *
Eu
Ts-V
Ts-P
0
40
80
120
CamKII
α
protein levels
(% of Eu)
A
C
DEF
GH
I
Eu
Ts-V
Ts-P
0
40
80
120
mean AMPA/GABA (%)
*
B
Eu
Ts-V
Ts-P
0
40
80
120
pGluA1
S845
/GluA1 ratio
(% of Eu )
J
Antioxidants 2023, 12, 111 16 of 27
dependent AMPA receptor trafficking by the mean of two phosphorylation sites, i.e., S845
and S831 [74–76]. S845 phosphorylation promotes GluA1 surface expression and increases
channel open-probability, while S831 phosphorylation augments the single-channel con-
ductance [74–76]. Our data show no differences for GluA1 protein levels (Figure 5D).
GluA1 S831 phosphorylation was significantly increased in Ts-V mice (+75% vs. Eu, p =
0.008), while it reduces to levels comparable to those observed in Eu mice after the
KYCCSRK administration in Ts-P mice (−60% vs. Ts-V, p = 0.03) (Figure 5E). No significant
changes for GluA1 S845 phosphorylation were observed (Figure 5F). CamKIIα is respon-
sible for GluA1 S831 phosphorylation and changes in CamKIIα activity contribute to the
turnover and modulation of GluA1 at the plasma membrane [77]. Our results show no
changes for CamKIIα protein levels (Figure 5G), while a consistent CamKIIα activation
(increased T236 phosphorylation) is observed in Ts-V mice (+294% vs. Eu, p = 0.004), that
is significantly reduced following KYCCSRK treatment (−196% vs. Ts-V, p = 0.03) in Ts-P
mice (Figure 5H). No changes for synaptophysin (pre-synaptic) and PSD95 (post-synap-
tic) were found (Figure 5I,J).
Overall, these data suggest that an imbalance between inhibitory vs. excitatory cur-
rents likely driven by an increased AMPA receptor conductance (triggered by CamKIIα-
mediated GluA1 S831 phosphorylation) can be observed in Ts2Cje mice frontal cortex,
while the KYCCRSK intranasal administration efficiently restores such alterations.
3.6. Correlation Analyses and PCA
The whole set of measured proteins was used to build a correlation matrix to look at
any significant association. Results are reported in Figure 6A,B. Among significant corre-
lations, we observed that IR activation is negatively associated with 3-NT (r= −0.58, p =
0.04); and CamKIIα activation (T236/CamKIIα, r= −0.90, p < 0.001) levels while IRS1 inhi-
bition (S307/IRS1) is positively associated with 3-NT (r= 0.64, p = 0.02). Furthermore,
among the proteins involved in AD neuropathology, APP-C99 levels are positively asso-
ciated with CamKIIα activation (r= 0.63, p = 0.03) while DYRK1A protein levels are posi-
tively associated with oxidative stress markers (HNE: r= 0.63, p = 0.02; 3-NT: r= 0.87, p <
0.001; PC: r= 0.64, p = 0.03), GluA1 S831 phosphorylation (S381/GluA1, r= 0.91, p < 0.001)
and CamKIIα activation (r= −0.54, p = 0.04). Morevoer, CamKIIα activation is positively
associated with GluA1 S831 phosphorylation (r= 0.57, p = 0.003). Then, the multivariate
data were arranged in a matrix which was then subjected to a principal component anal-
ysis (PCA). Resulting scores plot is displayed in Figure 7. PCA outcomes show how the
first principal component (PC1) differentiates between Ts-V (at positive scores) and Eu
and Ts-P mice (at negative scores), these latter sharing similar features. In particular, by
looking at the loadings (Figure 7A), it appears that Ts-V mice differ from Eu and Ts-P mice
because they are characterized by brain insulin resistance (lower pIR/IR and higher
pIRS1S307/IRS1, and pIRS1S636/IRS1), the accumulation of AD-associated neuropatho-
logical hallmarks (higher APP, APP-C83, APP-C99, DYRK1A, and TAU), increased oxida-
tive stress levels (higher PC, HNE and 3-NT) and the aberrant activation of proteins reg-
ulating synaptic plasticity mechanisms (higher pGluA1 S831, pCaMKIIα T286). Moreover,
it is possible to differentiate Eu from Ts-P mice along the second principal component
(PC2), since the former falls at negative scores and the latter at positive scores. In this case,
it is also possible to interpret the observed differences in terms of the measured variables,
by inspecting the loadings plot in Figure 7B. In particular, Ts-P mice are characterized by
an improvement of brain insulin signaling activation (higher pAKT/AKT), an ameliora-
tion of mitochondrial bioenergetics (higher CV/VDAC, CIII/VDAC, CII/VDAC, and
CI/VDAC) and an improvement of synaptic plasticity mechanisms (higher PSD95 and
Synaptophysin).
Antioxidants 2023, 12, 111 17 of 27
Figure 6. Correlation analyses. Pearson correlation analyses were performed to explore associations
among all the proteins measured in in our work. Correlations are shown as correlation matrix. In
(A) Pearson r values are reported. In (B) significant correlations are indicated.
IR
pIR/IR
IRS1
Y612/IRS1
S636/IRS1
S307/IRS1
AKT
pAKT/AKT
BVRA
PTEN
pPTEN/PTEN
APP
C83
C99
ADAM10
BACE1
TAU
p TAU-S404
p TAU-AT8
DYRK1A
HNE
3-NT
PC
VDAC
CI/VDAC
CII/VDAC
CIII/VDAC
CIV/VDAC
CV/VDAC
S831/GluA1
S845/GluA1
GluA1
T286/CaMKIα
CaMKIIα
PSD95
Synaptophysin
MTOR
pMTOR/MTOR
LC3 II/I
BECLIN
ATG 5
LAMP1
SQTM1
4EBP1
p4EBP1/4EBP1
0.01
0.02
0.03
0.04
0.05
Insulin signaling
AD hallmarks
Mitochondria
Synaptic proteins
Autophagy
Oxidative stress
Insulin
signaling
AD
hallmarks
Oxidative
stress
Mitochondria
Synaptic
proteins
Autophagy
Insulin
signaling
AD
hallmarks
Oxidative
stress
Mitochondria
Synaptic
proteins
Autophagy
A
B
Pearson r p value
IR
pIR/IR
IRS1
Y612/IRS1
S636/IRS1
S307/IRS1
AKT
pAKT/AKT
BVRA
PTEN
pPTEN/PTEN
APP
C83
C99
ADAM10
BACE1
TAU
p TAU-S404
p TAU-AT8
DYRK1A
HNE
3-NT
PC
VDAC
CI/VDAC
CII/VDAC
CIII/VDAC
CIV/VDAC
CV/VDAC
S831/GluA1
S845/GluA1
GluA1
T286/CaMKIα
CaMKIIα
PSD95
Synaptophysin
MTOR
pMTOR/MTOR
LC3 II/I
BECLIN
ATG5
LAMP1
SQTM1
4EBP1
p4EBP1/4EBP1
IR
pIR/IR
IRS1
Y612/IRS1
S636/IRS1
S307/IRS1
AKT
pAKT/AKT
BVRA
PTEN
pPTEN/PTEN
APP
C83
C99
ADAM10
BACE1
TAU
p TAU-S404
p TAU-AT8
DYRK1A
HNE
3-NT
PC
VDAC
CI/VDAC
CII/VDAC
CIII/VDAC
CIV/VDAC
CV/VDAC
S831/GluA1
S845/GluA1
GluA1
T286/CaMKIα
CaMKIIα
PSD95
Synaptophysin
MTOR
pMTOR/MTOR
LC3 II/I
BECLIN
ATG 5
LAMP1
SQTM1
4EBP1
p4EBP1/4EBP1
-1.0
-0.5
0
0.5
1.0
Antioxidants 2023, 12, 111 18 of 27
Figure 7. Principal component analysis results. Principal component analysis (PCA) was performed
on the results collected in mice to determine variance contribution of the components associated
with the observed neuroprotective effects promoted by KYCCSRK administration in Ts-P mice.
Score plots (A) and loadings (B) graphs are shown.
4. Discussion
Growing evidence suggests that brain insulin signaling, other than having a role in
the regulation of cerebral metabolism, regulates key molecular pathways involved in
mood, behavior, and cognition [48], that are impaired following development of brain
insulin resistance. In general, insulin resistance is defined as the reduced response to in-
sulin by target cells, due to reduced IR levels and/or activation or increased IRS1 inhibi-
tory phosphorylation that finally results in impaired glucose uptake and cell metabolism
[49–51]. Moreover, brain insulin resistance mediates synaptotoxic effects in several ways
leading to (1) the accumulation of AD neuropathological hallmarks [10,52–54]; and (2)
A
B
Ts-V
Ts-P
Antioxidants 2023, 12, 111 19 of 27
synapse loss, impaired autophagy, and increased neuronal apoptosis [10,55,56]. These ob-
servations collected in vitro and in animal models have been strengthened by clinical
studies reporting that the failure in brain energy metabolism responsible for the cognitive
decline during aging or AD could be driven by the development of brain insulin resistance
particularly at the early stages [10,57].
We previously reported on the accumulation of markers of brain insulin resistance,
such as reduced IR protein levels and increased IRS1 inhibition, in the brain of young DS
individuals before AD development [23]. Furthermore, we proposed that the develop-
ment of brain insulin resistance triggers AD onset in DS since reduced IR protein levels
along with the impairment of insulin signaling are associated with a great amyloidogenic
cleavage of APP (increased APP C99 levels) in DS < 40 years old but not in age-matched
controls [23].
Here we show for the first time that brain insulin signaling activation can be rescued
by means of the intranasal administration of the KYCCSRK peptide in Ts2Cje mice. Our
results demonstrate that KYCCSRK leads to IR activation along with increased activation
of IRS1 and downstream targets, i.e., AKT in the frontal cortex. The activation of brain
insulin signaling can be observed also in Eu mice, suggesting that the peptide is able to
exert its effects under physiological conditions. These results agree with the role proposed
for KYCCSRK promoting insulin-independent activation of IR and AKT in several cell
lines and to an extent like results observed with insulin stimulation [27,40] as well as in
vivo [41]. Furthermore, the observed increased activation of IR and AKT following
KYCCSRK administration in Ts2Cje mice strengthens the idea that such a peptide can
cross the BBB and reach the frontal cortex when administered through the intranasal
route.
Defects of insulin signaling have been shown to co-occur with Aβ plaques and Tau
phosphorylation in the temporal lobe, hippocampus, and cerebellum [14,18,52,58]. Brain
insulin resistance triggers the accumulation of APP toxic fragments (e.g., Aβ and -C99)
either by favoring an aberrant APP cleavage or by inhibiting their clearance [14–
16,53,59,60]. Increased APP-C83 and -C99 levels in human DS brains were observed to an
even greater extent than those found in AD brains [61]. Moreover, the accumulation of
both APP-C83 and APP-C99 was shown to contribute to endo-lysosomal abnormalities
and the buildup of other oxidized substrates that are responsible for damaging cellular
components in the DS brain [62–64]. Results of the current study show that KYCCSRK
peptide, by rescuing insulin signaling activation, decreases the levels of APP-CTFs frag-
ments (both -C83 and -C99) within the frontal cortex regardless of APP. APP-C83 levels
are reduced independently of ADAM10 protein levels, and possibly by increased C83
clearance following the KYCCSRK treatment. The improvement of insulin signaling
seems to have a role, in agreement with previous studies showing that insulin administra-
tion reduces APP CTFs levels both in vitro and in vivo [65,66]. Indeed, a strong positive
correlation between C83 and IRS1 inhibition (S307) was found in our study. We observed
that the levels of BACE1 protein—the secretase responsible for the generation of APP-C99
fragment in the amyloidogenic pathway [67]—are consistently decreased in the Ts2Cje
mice following KYCCSRK administration, which may explain reduced APP-C99 levels
observed in Ts-P mice. Furthermore, in a recent study, BACE1 was shown to impair IR
activation [68]. Hence, it might be hypothesized that reduced BACE1 protein levels also
contribute to the observed activation of IR in the frontal cortex following KYCCSRK ad-
ministration in Ts-P mice. Lower BACE1 protein levels seem to result from reduced tran-
scription (reduced mRNA levels). Whether the peptide on its own or the activation of in-
sulin signaling represses BACE1 gene transcription was not established in this paper, and
is out of the scope of this study. This is an intriguing aspect that deserves further investi-
gations and studies on the topic are ongoing in our laboratory.
Other than APP-associated neuropathology, KYCCSRK treatment reduced TAU
phosphorylation in the frontal cortex of Ts-P mice. This result is remarkable because it
underlies protective mechanisms of the KYCCSRK peptide in terms of TAU biology in DS.
Antioxidants 2023, 12, 111 20 of 27
Lack of differences between Eu and Ts-V mice in our study is not surprising, considering
that changes of TAU phosphorylation are both age- and brain region-specific due to the
genetics of the different DS mouse models used in the literature [69–71]. To better under-
stand the mechanisms responsible for reduced TAU phosphorylation, we looked at the
DYRK1A protein. DYRK1A is abundantly expressed in the brain and interacts with nu-
merous cytoskeletal, synaptic, and nuclear proteins in neurons, including TAU [72]. Inter-
estingly, the DYRK1A gene is triplicated in DS [73,74], having a role in the observed im-
pairment in neuronal development and neuronal activities [73,74]. Moreover, DYRK1A
protein through its kinase activity promotes TAU phosphorylation in DS [73,75].
KYCCSRK led to reduced DYRK1A protein levels in the frontal cortex of Ts-P mice. No-
tably, overexpression of DYRK1A in peripheral organs was related to diabetes pheno-
types [76], while pharmacological inhibition of DYRK1A leads to an improved glycemic
control in both mice and human cells [77–79]. Together, the current results and previous
observations further support the hypothesis that elevated DYRK1A protein levels might
have a role in brain insulin resistance development and AD pathological hallmarks accu-
mulation in DS.
KYCCSRK neuroprotective effects include reduced oxidative stress levels observed
in the frontal cortex of Ts-P mice. Previous studies demonstrated that oxidative stress is
an early event in DS [22,60,80,81], likely representing a key factor in the development of a
variety of pathological phenotypes. Furthermore, the overexpression of some HSA21
genes, e.g., APP and DYRK1A, and the dysregulation of gene/protein expression associ-
ated with the trisomy contribute to the increase of oxidative stress in DS [82–84], suggest-
ing that reduced APP amyloidogenic cleavage and DYRK1A protein levels might account
for the observed decrease of oxidative stress levels in Ts-P frontal cortex. The negative
correlations found between DYRK1A, and oxidative stress markers reinforce this hypoth-
esis.
The antioxidant properties of KYCCSRK also rely on the improved activation of in-
sulin signaling. Insulin signaling plays a pivotal role in the maintenance of mitochondrial
bioenergetics [85–87]. Conversely, the development of brain insulin resistance would pro-
mote mitochondrial dysfunctions responsible for reduced energy production, in turn, as-
sociated with an increase of reactive oxygen/nitrogen species formation, finally leading to
the oxidative/nitrosative damage of mitochondria as well as other cellular components
[86]. Brain insulin resistance and increased oxidative stress markers levels are tightly as-
sociated with cognitive dysfunctions [88–93] and also in DS [22,23,81], suggesting a close
link exists among these events. Intriguingly, insulin deprivation led to increased oxidative
stress levels in the mouse brain [94], while intranasal insulin treatment improved mito-
chondrial functions and reduced oxidative stress levels both in healthy mouse brains [94]
and in an AD mouse model [27]. In agreement with these findings, increased levels of
mitochondrial complexes were observed in Ts-P mice frontal cortex samples following
KYCCSRK administration, strengthening the role for improved insulin signaling activa-
tion in cell metabolism and energy production [8]. Indeed, our results highlight a robust
effect for CI and CIII levels, which are two complexes found significantly reduced in the
DS brain, playing a central role in mitochondrial dysfunction, energy production, and ox-
idative stress [82,95].
We underline that the neuroprotective effects elicited by the intranasal administra-
tion of the KYCCSRK peptide positively impact on brain plasticity, which is known to be
finely modulated by insulin [96]. In fact, insulin regulates synaptic plasticity mechanisms
by upregulating synaptic receptor subunits and SNARE proteins responsible for neuro-
transmitter release [48]. Conversely, alterations of insulin signaling in the central nervous
system impair brain plasticity, promote synapse loss and neurodegeneration, and accel-
erate brain aging [97]. Notably, GABAergic transmission is altered in DS, and several
studies suggest an excessive activity of inhibitory circuits in this condition [98]. In partic-
ular, DS mouse models show a plethora of alterations—including increased seizures inci-
dence, sleep alterations, and hyperactivity in locomotor behavior—that underlie an
Antioxidants 2023, 12, 111 21 of 27
imbalance between inhibitory vs. excitatory signals, likely due to an impairment of syn-
aptic inputs integration [99]. Our results agree with this hypothesis since an impairment
of the IAMPA/IGABA currents ratio in favor of excitatory currents (IAMPA) was observed in Ts-
V mice, while the KYCCSRK administration restored such alteration in Ts-P mice. Regu-
lation of AMPAR functions is highly dynamic in many different forms of synaptic plastic-
ity, including long-term potentiation and depression (LTP/LTD) and homeostatic synaptic
plasticity [100,101]. Mechanistically, restoration of the IAMPA/IGABA currents ratio seems to
be due to the reduced GluA1 S381 phosphorylation, which decreases GluA1 conductance
thus reducing AMPA currents [100,101] in Ts-P mice. GluA1 phosphorylation is regulated
by insulin signaling (reviewed in [102]). Notwithstanding, studies evaluating the effects
of exogenous insulin administration on AMPAR-mediated glutamatergic transmission
raised discordant conclusions showing either increased or reduced GluA1 phosphoryla-
tion (reviewed in [102]). We found strong positive correlations among DYRK1A,
pCamKIIα and pGluA1 S381 suggesting that reduced DYRK1A levels in Ts-P mice might
be responsible for reduced pCamKIIα-mediated GluA1 S381 phosphorylation. This is an
intriguing hypothesis because DYRK1A plays a pivotal role in synaptic plasticity mecha-
nisms [73,75] and particularly at the glutamatergic synapses [103]. In previous works from
Herault’s group, it has been shown that DYRK1A interacts with synaptic proteins, among
which CamKIIα—a kinase for GluA1 S831 residue [104] —was identified [103,105]. Over-
expression of DYRK1A was associated with increased CamKIIα protein levels in Dp1Yey
mice (a murine model for DS), that returned to normal following reducing DYRK1A gene
expression [103]. While the activation of CamKIIα was not evaluated in these studies, it is
conceivable to think that higher CamKIIα levels parallel higher CamKIIα activation in
Dp1Yey mice. We did not observe increased CamKIIα protein levels in the cortex of Ts-V
mice, but an increased CamKIIα activation, that to our opinion is responsible for increased
GluA1 S831 phosphorylation and impaired IAMPA/IGABA currents ratio in Ts-V mice. This
hypothesis is further reinforced by the observation that DYRK1A protein while interact-
ing with CamKIIα does not interact with GluA1 in wild-type mice brain extract [103].
Hence, overexpression of DYRK1A recruits synaptic proteins, e.g., CamKIIα, impairing
their activity in the DS brain, while normalizing DYRK1A gene expression and protein
levels is associated with neuroprotective effects [103,105,106].
Finally, we acknowledge that one limitation of our study is the relatively small group
of animals used to test the neuroprotective effects of the KYCCSRK peptide. However,
despite the sample size, consistent effects on outcome measures suggest this may be a
robust effect.
5. Conclusions
In conclusion, our work identified novel neuroprotective properties for the
KYCCSRK peptide that other than rescuing brain insulin signaling activation in Ts-P mice
also promotes an amelioration of some pathways involved in AD neuropathology devel-
opment (Figure 8). Moreover, the results of the current study further strengthen the idea
that the dysfunction of the insulin signalling pathway crosses with dysfunctions of pro-
teins encoded by genes on HSA21, e.g., DYRK1A and APP, thus contributing to worsening
a pre-existing condition defined on a genetic background in the DS brain. Hence,
KYCCSRK might represent a new therapeutic opportunity to restore the DS brain func-
tions affected by the above-mentioned alterations. This aspect is fascinating especially in
light of the role of the insulin signaling pathway in regulating energy metabolism and
cognitive functions and of the fact that accumulation of brain insulin resistance markers
is evident in children and adolescents with DS [20], and thus several years before AD
development. Finally, the significant effects obtained relative to DYRK1A and BACE1/C99
represent promising observations in searching for new molecules for ameliorating intel-
lectual disability and fighting AD development in DS.
Antioxidants 2023, 12, 111 22 of 27
Figure 8. Schematic representation of the molecular mechanisms associated with the neuroprotec-
tive effects mediated by the intranasal administration of the KYCCSRK peptide in Ts-P mice. Ar-
rows: stimulation; lines: inhibition.
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/antiox12010111/s1, Figure S1: Activation of brain insulin sig-
naling in response to increasing doses of KYCCSRK peptide administered through the intranasal
route in Eu mice; Figure S2: Reduced AD neuropathological hallmarks following KYCCSRK treat-
ment in primary cortical neurons isolated from Ts2Cje mice; Figure S3: The KYCCSRK peptide in-
tranasal administration in Ts2Cje mice does not promote changes of proteins regulating autophagy.
Author Contributions: Conceptualization, A.T., M.P. and E.B.; methodology, S.L., G.A., S.P., G.R,
P.C.; validation, A.T., S.L., G.A., S.P., G.R., P.C. and F.M.; formal analysis, A.T., C.R., E.P., C.G. and
E.B.; investigation, A.T., S.L., G.A., S.P., G.R., P.C. and F.M.; resources, E.B., C.R., E.P. and C.G.; data
curation, A.T., C.R., E.P., F.D.D., M.P. and E.B.; writing—original draft preparation, A.T., S.L., G.A.
and S.P.; writing—review and editing, C.R., E.P., C.G., F.D.D., M.P. and E.B.; supervision, E.B.; All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by Fondi Ateneo grant funded by Sapienza University n°
RM11715C77336E99 and n° RG11916B87F55459 to EB; n° RM11916B84D24429 and
RG12117A8697DCF1 to EP and GR. This project has received funding from the European Union’s
Horizon 2020 Research and Innovation Program under grant agreement No. 952455; and EpiEpiNet
to EP and GR. GR was supported by Italian Ministry of Health “Ricerca corrente”.
Institutional Review Board Statement: All the experiments were performed in strict compliance
with the Italian National Laws (DL 116/92), and the European Communities Council Directives
(86/609/EEC). The experimental protocol was approved by the Italian Ministry of Health
(#1183/2016-PR). All efforts were made to minimize the number of animals used in the study and
their suffering. Immediately after isolation, samples were put into liquid nitrogen and then stored
at −80 °C until utilization.
Informed Consent Statement: Not Applicable.
Data Availability Statement: Data are those available within the article and the supplementary
materials.
Antioxidants 2023, 12, 111 23 of 27
Conflicts of Interest: The authors declare no conflict of interest.
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