Galactosylated N-Glycans in the Choroid Plexus: A Possible Aging-Related Molecular Therapeutic Strategy for Alzheimer Disease

Abstract

Choroid Plexus (CP) and endothelium have important physiological and pathological roles in Alzheimer Disease (AD). The AD triple transgenic mouse model (3xTg-AD) is an animal model that mimics many critical hallmarks of the disease. Considering aging as the main AD risk factor, the AAV9 vector efficiency to carry out a non-invasive gene delivery in the CP of aged mice was studied. The results showed a greatly reduced GFP expression in the CP of aged mice compared to young mice. To explore the possible mechanisms involved in the
drastic reduction of GFP, possible AAV9 neutralizing factors were analysed in the mice-sera. There were no significant differences between young and old mice. The next step was to assess cell-surface N-linked glycans with terminal galactosyl residues since serve as the primary receptor for AAV9. We studied N-glycans in the CP of aged and 3xTg-AD mice by fluorescence staining using a biotinylated lectin and eNOS, a marker of endothelium. A decrease in terminally galactosilated N-glycans was also found in aged and 3xTg-AD mice. The results indicated that very reduced AAV9 tropism in the CP of aged mice correlates both
with a significant reduction in galactose residues of glycans that occurs in aging, as well as with a decrease of eNOS in endothelial cells. In order to increase AAV9 tropism in CP both a neuraminidase-based molecular therapeutic strategy and an improvement of endothelium function for AD is proposed.

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Citation: Brito-Armas JM, Chillón M, Méndez-Medina R, Bosch A and Castro-Fuentes R. Galactosylated
N-Glycans in the Choroid Plexus: A Possible Aging-Related Molecular Therapeutic Strategy for Alzheimer
Disease. Gerontol Geriatr Res. 2018; 4(1): 1034.
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Gerontology & Geriatrics: Research
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Abstract
Choroid Plexus (CP) and endothelium have important physiological and
pathological roles in Alzheimer Disease (AD). The AD triple transgenic mouse
model (3xTg-AD) is an animal model that mimics many critical hallmarks of the
disease. Considering aging as the main AD risk factor, the AAV9 vector efciency
to carry out a non-invasive gene delivery in the CP of aged mice was studied.
The results showed a greatly reduced GFP expression in the CP of aged mice
compared to young mice. To explore the possible mechanisms involved in the
drastic reduction of GFP, possible AAV9 neutralizing factors were analysed in
the mice-sera. There were no signicant differences between young and old
mice. The next step was to assess cell-surface N-linked glycans with terminal
galactosyl residues since serve as the primary receptor for AAV9. We studied
N-glycans in the CP of aged and 3xTg-AD mice by uorescence staining using a
biotinylated lectin and eNOS, a marker of endothelium. A decrease in terminally
galactosilated N-glycans was also found in aged and 3xTg-AD mice. The results
indicated that very reduced AAV9 tropism in the CP of aged mice correlates both
with a signicant reduction in galactose residues of glycans that occurs in aging,
as well as with a decrease of eNOS in endothelial cells. In order to increase
AAV9 tropism in CP both a neuraminidase-based molecular therapeutic strategy
and an improvement of endothelium function for AD is proposed.
Keywords: AAV9; Aging; Alzheimer disease; Choroid plexus; Galactosylated
N-glycans; Endothelium
models [7,8] and humans [9,10]. Besides there are clear evidences that
endothelial dysfunction is primarily responsible for the pathogenesis
that underlies Alzheimer disease [11].
e AD triple-transgenic mouse model (3xTg-AD) at 16 month-
old mimics critical hallmarks of the human disease: Aβ plaques and
neurobrillary tangles with a temporal- and regional- specic prole
[12]. However, it is little known how CP dysfunction in aging can
upset illness. In order to prevent AD-like pathology, our major goal
is to restore the CP dysfunction in 3xTg-AD mice through the use of
gene therapy, using as vector the non-invasive adeno-associated virus
serotype 9 (AAV9). AAV9 has a high tropism in CP epithelial cells
from young mice [13], due to the large existence of primary receptors
of AAV9, consisting in membrane glycans with terminal galactose
[14]. e problem is that the most studies are performed in young
models, and aging, the main risk factor in AD, is scarcely included in
the studies of this disease. e CP also presents age-related changes
[15,16]. Specically, aged CP epithelial cells present a general atrophy,
and consequently Cerebrospinal Fluid (CSF) production diminishes,
but also clearance of CSF out of the brain is delayed [17]. Nonetheless
there are few studies that combine both molecular pathologies of AD
and aging, as well as very few gene delivery preclinical studies in old
animals.
In this work we analysed both how aging may aect AAV9
tropism in CP and others brain regions, as well as possible underlying
Introduction
Alzheimer’s Disease (AD) is the most common form of
neurodegenerative dementia in the elderly. e pathological features
that characterize AD are neuronal atrophy, synapse loss and the
progressive accumulation of senile plaques. ese plaques are
composed of various Amyloid Beta (Aβ) peptides, including the 40
and 42 amino acid cleavage products (Aβ₄₀ and Aβ₄₂) of the amyloid
precursor protein, and intracellular Neurobrillary Tangles (NFTs),
containing hyperphosphorylated tau protein [1,2]. is accumulation
in AD is quite toxic for neurons mostly in cortex and hippocampus
[3], however increasing data for animals support the notion that
compromised function of Choroid Plexus (CP) and defective
cerebrospinal uid production and turnover with diminished
clearance of the Aβ peptides normally produced in brain, may be a
mechanism implicated in the exacerbation of AD [4]. Additionally,
it has been suggested that the accumulation of Aβ in CP further
increases even more the disruption of the blood-cerebrospinal uid
barrier [5,6], which may impact on neurodegeneration. Currently,
little is known about transport and metabolic responses of CP to the
disrupted homeostasis of Aβ in AD.
Although CP participates in the development of AD, another
important factor in the clearance of Aβ is the vascular system. ere
are various authors describe that the increase of oxidative Reactive
Oxygen Species (ROS) lead endothelial dysfunction both animal
Special Article - Alzheimer’s Disease
Galactosylated N-Glycans in the Choroid Plexus: A
Possible Aging-Related Molecular Therapeutic Strategy
for Alzheimer Disease
Brito-Armas JM1, Chillón M2, Méndez-Medina R3,
Bosch A2 and Castro-Fuentes R3*
1Research Unit, University Hospital of the Canary Islands,
Spain
2Department of Biochemistry and Molecular Biology,
Universitat Autònoma de Barcelona, Spain
3Department of Basic Medical Sciences, University of La
Laguna, Spain
*Corresponding author: Rafael Castro-Fuentes,
Department of Basic Medical Sciences, School of Health
Sciences, Section Medicine, University of La Laguna,
38200 La Laguna, Tenerife, Spain
Received: September 28, 2017; Accepted: February 12,
2018; Published: February 19, 2018

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mechanisms. In addition, we studied the involvement of such
mechanisms in a triple transgenic mouse model of Alzheimer’s
disease.
Materials and Methods
Animals
3xTg-AD mice harboring three mutant genes: beta-APP
(APPswe), presenilin-1 (PS-1M146V) and tauP301L, and the
corresponding wild type mice, were provided by Dr. Lydia Giménez-
Llort (Autonomous University of Barcelona, Spain). Eight 3xTg-AD
mice and eight non-transgenic control mice (Non-Tg), 16 month-old,
were used. For studies of aging, we used 6-week-old and 22-month-
old C57BL/6j male mice. All animal experiments were approved by
the bioethical committee of the University of La Laguna (Reference
# 091/010) and are in accordance with the European Communities
Council Directive of 22 September 2010 (2010/63/EU) regarding the
care and use of animals for experimental procedures.
AAV9-mediated gene delivery experiments
Construction and production of adeno-associated vector
AAV9 vectors were synthesized at the Vector Production Unit of
the Autonomous University of Barcelona using the triple transient
transfection method with Polyethyleneimine (PEI). eGFP were
used as reporter gene. HEK293 cells were harvested 72 hours aer
transfection and the viral particles were puried by ultracentrifugation
on an iodixanol gradient according to the Zolotukhin et al. [18]
method. We injected both groups of C57BL/6j mice with AAV9-
eGFP intravenously at a dose of 1,3x1013 viral genomes per kg in 100µl
PBS (n=8 per group). is viral genome dose was chosen to avoid
side eects as far as possible [18]. At 6 weeks post-injection mice were
killed and subjected to either morphological analysis or molecular
biology and biochemical studies.
Western-blotting, Immunouorescence and lectin-
staining
Proteins were isolated with M-PER extraction reagent (Pierce).
Protein sample concentrations were equalized by the bicinchoninic
acid method and separated by SDS-PAGE and transferred to a
nitrocellulose membrane. e blotted membranes were incubated
with anti-GFP antibodies (1: 50000; SySy). α-tubulin was used as
Figure 1: AAV9-mediated eGFP expression was greatly reduced in epithelial cells of the CP in old mice respect to young mice.
A. A representative confocal microscopy image of the CP stained with antibodies anti-eGFP (green) anti-transthyretin (red) and the merge from young mice (6
weeks old) and old mice (22 months old) is shown. Scale bars, 50μm.
B. Western blot for eGFP was carried out for quantifying the protein levels. n = 4 animals per group; **p<0.001 Old vs. Young mice (t -test two tailed).
Figure 2: Neutralizing factors do not explain the poor AAV9 transduction in CP from old mice.
A. Percent inhibition of transduction of AAV9 neutralized with mice sera dilutions. No signicant changes were detected between sera of young and old mice (n=5,
sera of non-AAV9 treated animals were also included as control).
B. ELISA-IgG values against AAV9 in serum of young and old mice are shown (n=5, sera of non-AAV9 treated animals were also included as control). Serum IgG
levels of young and old mice are signicantly different. *p<0.05 (t -test two tailed).

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a loading control and Chemidoc-Quantity-One soware (Biorad
Laboratories) was used to perform quantitative analyses. For
immunouorescence, the sections of xed tissue were incubated with
anti-GFP antibody (1: 1500; SySy), eNOS (1:1000; Sigma) and anti-
transthyretin (1:200; DAKO). For studying the primary receptor of
AAV9 we used the Erythrina cristagalli lectin (ECL) whose binds
to the galactose residues of cell-surface glycans [14]. Staining was
performed using this biotinylated lectin ECL (Vector Laboratories,
Burlingame, CA, USA) to 1:200. Lectin was visualized using
Streptavidin-CY3 or Streptavidin-CY2 (Vector Lab, Burlingame,
CA; 1:600). e uorescence images were taken with a confocal
microscope Olympus Fluoview FV10 (Olympus FV10-ASW2Q,
Shinjuku, Tokyo, Japan).
Neutralizing factors
To test for the prevalence of neutralizing factors against AAV9 in
the sera of the mice, AAV2/9-CMV-luciferase was incubated at 37ºC
for 30 minutes with non-heat-inactivated serum samples diluted in
infection medium (DMEM+2% FBS+1% PenStrep) to 1:50, 1:150 and
1:450. Luciferase activity was detected by adding luciferin substrate
(Pierce Firey Luciferase Flash Assay kit) and reading the resulting
luminescence in VICTOR 3 (Perkin Elmer). Total concentration
of IgG antibodies against AAV9 present in the animal sera was
determined by ELISA.
Statistical analysis
Data were reported as Means ± Standard Error (SEM).Statistical
comparisons were performed using unpaired two-tailed Student’s
t-tests or Mann-Whitney U-test. Dierences were considered to be
signicant when P≤0.05.
Results and Discussion
Our study supports the ability of AAV9 to successfully traverse
the vasculature and eciently transducing CP of young mice, aer
intravenous administration of a marker gene (eGFP). However, we
have found that this capacity decreases dramatically in several brain
regions in 22 months old mice: Regarding CP, aer performing
immunouorescence of eGFP and transthyretin, we observed a
drastic reduction in the expression of eGFP in old mice compared
to young mice (Figure 1A). ese data were corroborated with the
studies of immunoblotting where we also observed a reduction of
eGFP levels in CP from old mice compared to young mice (Figure
1B).We found also a highly reduction of eGFP levels in cortex and
hippocampus from old- compared to young mice (Supplementary
Figure 3: A reduced AAV9-primary receptor expression is observed both in CP from old- versus young mice and in 3xTg-AD versus Non-Tg mice.
A. ECL lectin (red) was used to detect N-glycans containing terminally β1,4-galactose (primary receptor of AAV9). n = 4 animals per group; ***p<0.0001 (Mann
Whitney test) Old- vs young mice. Scale bars, 50μm.
B. The primary receptor of AAV9 also decreased in the CP from the 3xTg-AD mice respect to non-transgenic mice (green). n = 4 animals per group; **p <0,001
(Mann Whitney test) 3xTg-AD vs Non-Tg mice. Scale bars, 50μm.
Figure 4: Decreased expression of eNOS in brain regions from old- versus young mice.
A. Representative image of eNOS immunouorescence in vascular endothelium from different brain regions (cortex, hippocampus and choroid plexus).
B. Quantication of the eNOS mean intensity of uorescence in vessels from different brain regions (except choroid plexus) and quantication of the eNOS mean
intensity of uorescence in vascular endothelium of choroid plexus (n = 4 per group); ***p<0.0001 old versus young mice (Mann-Whitney U test).

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Figure 1).
e much reduced eGFP expression could be due to AAV9
neutralizing factors in serum of aged mice [19]. To ensure that AAV9
there arrived to CP, we evaluated both the neutralizing antibodies
and other possible neutralizing factors in mouse sera which could
prevent the arrival of AAV9 to CP. However, there were no signicant
changes both in the neutralizing factors (Figure 2A) as in IgG against
AAV9, and even IgG was signicantly lower in old-mice serum than
in young mice (Figure 2B). is phenomenon could be due to the
immunosenescence described as age advances in all species [20]. In
mice, the strongest immune system is detected between 6 weeks and
6 months, while at 23 months the robustness of the immune response
is much lower. However, we cannot discard the hypothesis that lower
neutralizing factors and IgG titers in old animals could be due to
the reduced level of transduction in this group of animals, leading
to signicantly less antigen-presenting cells and thus, to poorer
activation of the immune system.
Once discarded neutralizing factors or possible antibodies against
AAV9, since there was not statistical dierences between serum from
young- and old mice (Figure 2), the next step was to study the primary
receptor of AAV9 in CP. ECL staining revealed that the terminal
galactosylation of cell surface glycans was signicantly reduced in
CP from old mice compared to youngs (Figure 3A), it suggesting one
possible mechanism of reduced eciency transduction of AAV9 in
CP from old mice. Moreover, ECL staining showed that the terminal
galactosylation of cell surface glycans was also reduced in CP of
3xTg-AD mice respect to Non-Tg mice (Figure 3B). is could be a
drawback for a future AAV9-mediated gene therapy targeted choroid
plexus of 3xTg-AD mice. It has been described a correlation between
agalactosylated IgG and aging human, and also in old-mice models
[21]. Our data conrm this change of glycan prole, but in a very
important structure for AD as CP. Many researchers think that the
agalactosylated glycans are a good marker of aging; even in serum of
patients with dementia these molecules are found a signicant lower
levels than controls [22], which could also be an excellent pathologic
marker for AD.
Nevertheless, according to our results, the primary receptor
amount only was 50% reduced in the old mice so that other factors
could be inuencing in the poor entrance of AAV9. Since the blood
ow is lower in aging and Alzheimer disease [23], next step was to
analyze eNOS immunouorescence in dierent vessels of cortex,
hippocampus and the CP. We found a signicant reduction of eNOS
in old mice respect young’s (Figure 4). ese data support that there is
a reduction in blood ow from old mice, which together to the fall of
the primary receptor could explain the almost null tropism of AAV9.
However, these facts, which a priori could be negative, open a range of
possibilities to improve the AAV9 tropism. One of them, proposed by
Bell et al. (2011), consisted in using Neuraminidase (NA) to increase
the abundance of the receptor on target cells, improving both the
vector ecacy and delivering of AAV vectors to their therapeutic
targets [24]. β-1,4galactose is the commonly observed penultimate
monosaccharide on most sialic acid-rich glycans [24], and NA is
able to remove the sialic acid, and display the primary receptor of
AAV9. However, it is not clear the role of both sialic acid-glycans
as galactose-terminal glycans on aging and it would be advisable to
perform more studies to know how it would aect the use of NA at
a systemic level. On the other hand, one of the best restorers of the
vascular system is the physical exercise, which produces angiogenesis
independent of aging [25,26]. e increase of blood vessel might
enhance the amount of viral particles of AAV9 that arrive at the CP,
and improve the tropism in AD.
erefore, we suggest that both a neuraminidase-based molecular
therapeutic strategy as an increase the angiogenesis through physical
exercise might increase the AAV9 tropism in the CP for AD. ese
results may help to gain further insight in using AAV9 to repair the
CP dysfunction described in 3xTg-AD mice.
Conclusion
An important challenge is to nd ways to selectively preventing
or minimize CP damage and CSF functions both in aging and in
states of neurodegeneration. Our results strongly suggest that AAV9
may be targeted noninvasively to CP, by using promoters of genes
selectively expressed, to restore its function in AD.
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Citation: Brito-Armas JM, Chillón M, Méndez-Medina R, Bosch A and Castro-Fuentes R. Galactosylated
N-Glycans in the Choroid Plexus: A Possible Aging-Related Molecular Therapeutic Strategy for Alzheimer
Disease. Gerontol Geriatr Res. 2018; 4(1): 1034.
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