[email protected]

CHAPTER
Plasticity as a therapeutic
target for improving
cognition and behavior in
Down syndrome
9
Carmen Martı́nez Cuea, Mara Dierssenb,*
a
Department of Physiology and Pharmacology, School of Medicine, University of Cantabria,
Santander, Spain
b
Cellular and Systems Neurobiology, Systems Biology Program, Centre for Genomic
Regulation (CRG), Barcelona Institute of Science and Technology, Universitat Pompeu
Fabra (UPF), Barcelona, Spain
*Corresponding author: Tel.: 933160140, e-mail address: [email protected]
Abstract
Early intervention and environmental optimization have been central to management of Down
syndrome (DS) and much of current treatment is still focused in strategies that involve early
education plans. This approach has provided significant improvements for Down syndrome
but it is not providing a full success.
The discovery of an increasing number of genes and molecular pathways linked to intellectual disability and involving a range of synaptic and plasticity-related mechanisms has open
new treatment opportunities that focus on targeted treatments boosting neural plasticity. We
here discuss some of these approaches, focusing on the effects of environmental enrichment
and on the discovery of pharmacological therapies showing beneficial effects even in some
clinical trials in adult individuals with Down syndrome. Targeting plasticity impairments
in DS is thus a promising strategy to promote cellular mechanisms involved in learning
and memory within key cognitive brain region and could lead to improved connectivity.
Keywords
Neuronal plasticity, Environmental enrichments, Epigenetics, EGCG, Environ-mimetic drugs
1 Introduction
For decades, efforts to promote cognitive therapies in DS have been regarded with
skepticism for finding any true therapeutic benefit, and with reluctance to invest the
time and resources into low-gain clinical trials. The most important reason is that the
Progress in Brain Research, Volume 251, ISSN 0079-6123, https://doi.org/10.1016/bs.pbr.2019.11.001
© 2020 Elsevier B.V. All rights reserved.
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putative mechanism(s) of intellectual impairment specific to trisomy 21 have never
been seen as targetable because of the ballast of the developmental effects on the
brain. This is also the case for many neurodevelopmental disorders such as autism,
epilepsy and other forms of learning disabilities all of which share some form of synaptic dysfunction, with a myriad of variations in synaptic structure and function leading to similar learning and memory deficits. These forms of synaptic dysfunction
include variations in synaptic proteins, such as changes in receptors for glutamate,
GABA or other neurotransmitters, and impaired function at various sites and mechanisms of the synapse. They most probably lead to an inability to adequately respond
to the environmental requirements, called activity-dependent plasticity. The consequences can be found in changes in the physical structure of the synapse, as reflected
by abnormalities in spine morphology and density, that adversely affect the information processing efficiency and storage capacity of neural networks (BenavidesPiccione et al., 2004; Dierssen and Ramakers, 2006; Morè et al., 2019).
The neurocognitive phenotype of DS stems from deficits in “late-developing”
neural systems, with greater gray matter reductions in the frontal lobe, hippocampus
and cerebellum (Lott and Dierssen, 2010; Menghini et al., 2011), areas that have
major roles in the DS-specific mnesic alterations (Krasuski et al., 2002; Teipel
et al., 2004). Those are regions characterized by a rich neuronal connectivity, based
predominantly on excitatory connections on the dendritic spines of pyramidal neurons and show the highest plasticity in the brain.
In this chapter we focus on some successful strategies increasing neurogenesis,
and promoting dendritic rewiring via pro-cognitive treatments, which could provide
a unique opportunity to ameliorate neuronal morphology, wiring, and cognitive
function.
2 Neurogenesis enhancers
During the entire life span of many animals, including mice, cell proliferation takes
place in the subventricular (SVZ) zone and in the subgranular zone (SGZ) of the
dentate gyrus (DG).
Neurogenesis is severely compromised in DS from early developmental stages.
In DS fetuses, a reduced number of dividing cells are found in the DG, lateral ventricle (Contestabile et al., 2007; Guidi et al., 2008), external granular layer (EGL) of
the cerebellum and in the ventricular zone (VZ; Guidi et al., 2011) (see chapter
“Translational validity and implications of pharmacotherapies in preclinical models
of Down syndrome” by Martinez Cue). Reduced proliferation of neural precursor
cells is also found in mouse models of DS (i.e., Ts65Dn, Ts1Cje and Ts2Cje) during
embryonic and perinatal stages in the neocortex, in the subventricular zone (SVZ)
and cerebellar EGL (Chakrabarti et al., 2007; Hewitt et al., 2010; Ishihara et al.,
2010; Laffaire et al., 2009; Moldrich et al., 2009) and from birth to adulthood in
the hippocampal DG (Bianchi et al., 2010a; Contestabile et al., 2007; Lorenzi and
Reeves, 2006; Trazzi et al., 2011). These alterations in neurogenesis depend on
impaired neuronal precursor proliferation, slowing of the cell cycle and altered
2 Neurogenesis enhancers
differentiation (Bahn et al., 2002; Esposito et al., 2008; Hewitt et al., 2010; Laffaire
et al., 2009; Moldrich et al., 2009). However, more recent studies conclude that the
balance across different process is what defines the final neuronal number. For example, although adult Ts65Dn mice have a lower number of proliferating cells it is
compensated by reduced cell death, thus leading to a final number of mature cells
similar to controls (Pons-Espinal et al., 2013). Therefore, reduction of adult neurogenesis by itself would not be responsible for the neuronal hypocellularity in the hippocampus (López-Hidalgo et al., 2016) in this model. Even so, if defects in adult
neurogenesis in DS were partially responsible for their cognitive alterations, therapies targeted to rescue neurogenesis could be a good approach to treat intellectual
disability in DS individuals.
There is a wide body of literature that reports different pharmacotherapies that
rescue neurogenesis and cognitive deficits in murine models of DS when administered in pre or in postnatal stages. Most of them do not target DS-specific mechanisms, but are explored due to their general neurogenic properties. One example
is fluoxetine, a selective serotonin reuptake inhibitor antidepressant that has been
shown to increase neurogenesis in mouse (Malberg et al., 2000). In Ts65Dn mice,
chronic treatment during prenatal (Guidi et al., 2014), early postnatal (Bianchi
et al., 2010a; Stagni et al., 2015) and adult stages (Clark et al., 2006) restored neurogenesis and cognition. Similarly, lithium, a drug prescribed for bipolar depression
also rescued neurogenesis in Ts65Dn mice DG at 6 months of age (Contestabile
et al., 2013) and in the SVZ at 12-months of age (Bianchi et al., 2010b). Because
both fluoxetine and lithium are approved for other indications in humans they could
be easier to translate into clinical trials. In fact, a clinical trial is ongoing (Stella Maris
Foundation; EudraCT Number: 2011-001556-11), though no results are yet reported.
However, possible side effects in neonates upon prenatal treatment with antidepressants cannot be ruled out. Fluoxetine has been associated with risk of specific cardiovascular malformations (Reefhuis et al., 2015) although not all studies find a
substantial teratogenic effect of SSRI (Furu et al., 2015). However, the potential risk
of preterm birth (Hayes et al., 2012) and pulmonary hypertension in the neonate
(Chambers et al., 2006; Olivier et al., 2013) may occur, and in utero exposure to serotonin reuptake inhibitors may result in a neonatal withdrawal syndrome (MosesKolko et al., 2005; Sanz et al., 2005). In any case, there are no published data on
DS babies born from mothers taking fluoxetine or other antidepressants and a pilot
feasibility trial of perinatal fluoxetine treatment proposed at the Southwestern Medical Center of the University of Texas in 2014 (Byerly, M., Carlin, M., HorsagerBoehrer, R., 2014. A pilot feasibility trial of prenatal and early postnatal fluoxetine
treatment for intellectual impairments of Down syndrome) has not yet reported beneficial effects.
Increasing evidence indicates that adult hippocampal neurogenesis is implicated
in the establishment of long-term potentiation (LTP) and has a role in hippocampaldependent learning and memory (Malberg et al., 2000; Shors et al., 2002). A study
employing a CNTF- or BDNF-based prenatal to early postnatal pharmacotherapy in
the Ts65Dn mouse model showed beneficial effect both on neurodevelopment and
on cognition in adult life (Kazim et al., 2017). In the same direction, a flavone
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derivative, 7,8-dihydroxyflavone (7,8-DHF), a small molecule that crosses the
blood–brain barrier and binds with high affinity and specificity to the TrkB receptor,
subcutaneously injected with 7,8-DHF in P3-P15 Ts65Dn pups exhibited a large increase in the number of neural precursor cells in the DG and restoration of granule
cell number, density of dendritic spines and levels of the presynaptic protein synaptophysin, indicating that the recovery of the hippocampal anatomy translated into a
functional rescue (Stagni et al., 2017). Another example is pharmacological blockade of cannabinoid receptor CB1R, which restores memory deficits, hippocampal
synaptic plasticity and adult neurogenesis in the subgranular zone of the DG
(Navarro-Romero et al., 2019).
Other drugs that have been demonstrated to restore neurogenesis in prenatal and/
or early postnatal treatment studies and improve cognitive deficits in Ts65Dn or
other DS mouse models are, among others: the natural flavonoid luteolin (Zhou
et al., 2019), the flavonoid agonist of the TRKB receptor for BDNF 7,8dihydroxyflavone (7,8-DHF) (Stagni et al., 2017), a BDNF-mimetic compound
(Parrini et al., 2017), corn oil (Giacomini et al., 2018), maternal choline supplementation (Velazquez et al., 2013), APP gamma-secretase inhibitors (Giacomini et al.,
2015), melatonin (Corrales et al., 2014), the long-acting β2 agonist formoterol
(Dang et al., 2014) and the GABAA α5 receptor negative allosteric modulator
RO4938581 (Martı́nez-Cue et al., 2013). Some of these are natural compounds like
melatonin or corn oil, the antioxidant α-tocopherol (vitamin E) (Shichiri et al., 2011),
the dietary supplement choline (Ash et al., 2014; Kelley et al., 2016; Moon et al.,
2010) that could be used safely in humans and thus, have potential for their use
in clinics. However, other drugs that have been proven to increase neurogenesis
and cognition in animal models of DS could not be used in humans because of their
intolerable side effects. One example is the synthetic activator of Sonic hedgehog
(Shh) pathway (Das et al., 2013) SAG 1.1. Treatment to Ts65Dn mice with SAG
1.1, increased mitosis, restored cerebellar granule cell precursor populations
(Roper et al., 2006) and rescued proliferation in the SVZ and DG (Trazzi et al.,
2011). In addition, administration of SAG 1.1 to newborn Ts65Dn mice restored cognition in these mice when they became adults (Das et al., 2013). However, because
SAG 1.1 is a very potent mitogenic, it could be carcinogenic, thus, precluding its use
in the human population. Finally other agents, such as active peptide fragments of
activity-dependent neuroprotective protein (ADNP) and activity-dependent neurotrophic factor (ADNF), two neuroprotective peptides, have also been assayed in
DS mouse models. ADNP and ADNF are released by glial cells in the brain and
are regulated by vasoactive intestinal peptide and they prevented developmental delay in the Ts65Dn mouse model of DS (Vink et al., 2009). However, it is not knows
whether they increase or restore neurogenesis.
A number of trisomic HSA21 genes have been proposed to play a role in these phenotypes, including Dyrk1A (Guimera et al., 1999; H€ammerle et al., 2003; Kentrup
et al., 1996; Yabut et al., 2010; Yang et al., 2001), Olig1 and Olig2 (Chakrabarti
et al., 2010; Lu et al., 2002; Takebayashi et al., 2000; Zhou and Anderson, 2002)
and App (Trazzi et al., 2011). In principle, it could be argued that their dosage normalization could also restore neurogenesis. In fact, the inhibitors of the activity of
3 Dendritic remodelers in Down syndrome
DYRK1A, ALGERNON, (Nakano-Kobayashi et al., 2017) and epigallocatechin
gallate (EGCG) (Stagni et al., 2016) promote neurogenesis in trisomic mice.
One important caveat regarding the consideration of neurogenesis as a putative
therapeutic target for DS adults is the controversial results about the actual existence
of adult neurogenesis in humans. Early studies reporting adult neurogenesis in the
human DG (Eriksson et al., 1998; Knoth et al., 2010; Roy et al., 2000; Spalding
et al., 2013) have been questioned by a recent report that could not detect neurogenesis in the human DG after the second year of life (Sorrells et al., 2018). Conversely,
two studies have demonstrated neurogenesis in the hippocampus of healthy humans
up to the eighth (Boldrini et al., 2018) and ninth (Moreno-Jimenez et al., 2019) decade of life. Moreno-Jimenez et al. also reported that the number of newborn neurons
progressively declined as AD advanced.
In summary, although there are numerous promising therapeutic strategies that
increase the cognitive abilities of TS mice through enhancing neurogenesis, only
continuous cognitive and physical stimulation are currently used in clinics, and have
been proved to be effective in the DS population. It remains to be demonstrated
whether drugs that enhance neurogenesis and cognition in mouse models of DS could
be useful in the DS population.
3 Dendritic remodelers in Down syndrome: The case of
epigallocatechin-3-gallate
In DS, the assumption is that deficits in activity-dependent plasticity lead to abnormal dendritic arborization that in turn would impact on a neuron’s function and
network connectivity and potentially contribute to learning and memory deficits
(Dierssen, 2012). Similar alterations have been linked to other neurodevelopmental
disorders, but are also a dominant feature of neurodegenerative diseases, suggesting
possible converging mechanisms. In the field of intellectual disability, the correction
of the dendritic phenotype has been used as readout of treatment efficacy and, in fact,
dendritic spine remodeling is usually associated to cognitive improvements in mouse
models. As such, many of the drugs mentioned above to increase neurogenesis, also
affect dendritic spine density. In this chapter we will deserve more attention to the
flavonoid epigallocatechin-3-gallate (EGCG), because it is one of the few molecules
that has demonstrated positive, though sub-clinic, pro-cognitive effects in a phase II
clinical trial in adults with DS (De la Torre et al., 2016). Green tea extracts contain a
number of catechins, including, EGCG, epigallocatechin (EGC), epicatechin-gallate
(ECG), and epicatechin (EC). Profiling catechins in human plasma and urine after tea
consumption showed that absorption, excretion, and metabolism of catechins affect
the bioavailability and potency, leading to differential bioactivities (Fung et al.,
2013; Lee et al., 1995).
EGCG is the most abundant and potent catechin extracted from green tea. It
crosses both the blood–brain barrier (Lin et al., 2007) and the placental barrier in
gestating rats (Chu et al., 2007). EGCG has a plethora of different effects, and
has thus been investigated in studies from various research areas, including cancer
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(Scarpa and Ninfali, 2015) and neurodegenerative disorders. Based upon its chemical structure, EGCG is classified as an antioxidant, which would counteract
increased oxidative stress resulting from the over activity of CuZnSOD1 in DS. It
also inhibits the transformation of nitrate and peroxynitrite into nitric oxide and
the microglia-mediated inflammatory response, thus decreasing neuronal damage
(Zhou et al., 2018). Other direct actions of EGCG are independent from antioxidative
mechanisms. EGCG interacts with proteins and phospholipids in the plasma
membrane and regulates signal transduction pathways, transcription factors, DNA
methylation, mitochondrial function and phosphorylation and autophagy (Shi
et al., 2018), to exert many of its beneficial biological actions. In vitro EGCG affects
a wide array of neuronal signal transduction pathways involved in plasticity, including JAK/STAT, MAPK, PI3K/AKT, WNT and NOTCH (Singh et al., 2011) (Fig. 1).
FIG. 1
Mechanisms of action of EGCG. Among others, EGCG targets include cell cycle proteins,
protein kinases, transcription factor, antiapoptotic proteins, growth factors and apoptotic
proteins.
Adapted from Singh, B.N., Shankar, S., Srivastava, R.K., 2011. Green tea catechin, epigallocatechin-3-gallate
(EGCG): mechanisms, perspectives and clinical applications. Biochem. Pharmacol. 82, 1807–1821.
3 Dendritic remodelers in Down syndrome
A recent paper suggested that EGCG could be beneficial through its capability to
inhibit metalloproteinase 9 (MMP-9). In DS, the metabolic pathway of nerve growth
factor (NGF) is altered due to deregulation of proteolytic enzymes such as MMP-9
and the plasminogen activation system, so that EGCG would protect NGF from
degradation acting on MMP-9, but also theaflavin inactivates PAI-1, which inhibits
tPA via PI3K/AKT, and MAPK (Wyganowska-Świa˛tkowska et al., 2018).
EGCG also interferes with epigenetic mechanisms at various levels, affecting the
chromatin opening state. It inhibits both DNA methyltransferases (Fang et al., 2003)
and class I histone deacetylases (HDAC 1,2,3,8) (Saldanha et al., 2014; Thakur et al.,
2012), it reduces the level of H327me3 and H2AK119 ubiquitination by reducing
polycomb protein levels (Choudhury et al., 2011), and affects miRNAs expression
(Milenkovic et al., 2012). As an example, EGCG increases methylation of alphasynuclein (SNCA) promoter proximal CpG sites and expression in Ts65Dn mice
(Ramakrishna et al., 2016). The property of EGCG of modulating epigenetic changes
makes it an ideal candidate for the treatment of DS, as its widespread epigenetic
effect might re-establish the lost epigenetic balance (De Toma et al., 2016). DS
individuals develop early in life Alzheimer’s disease neuropathology, in part due
to APP overexpression.
In the hippocampus of Alzheimer’s disease (AD) models, EGCG treatment
normalizes the expression of synaptic proteins (Guo et al., 2017; Xicota et al.,
2017). Strikingly, EGCG is also effective in other neurodegenerative disorders, such
as Parkinson’s disease (Singh et al., 2016). EGCG decreases beta-amyloid levels and
plaques via ADAM10-mediated promotion of the alpha-secretase proteolytic pathway,
and modulates tau-profiles leading to cognitive improvements (Rezai-Zadeh et al.,
2008). This effect is mediated via an estrogen receptor-a (ERa)/phosphoinositide
3-kinase/Ak-transforming dependent mechanism (Fernandez et al., 2010). EGCG also
conjugates directly with not folded natural peptides to inhibit the formation of toxic
intermediate products of α-synaptic nucleoprotein and amyloid protein and form a
nontoxic and disordered oligomer of these two proteins (Ehrnhoefer et al., 2008).
The heterogeneous effects of EGCG make it difficult to fully identify and understand the underlying molecular therapeutic mechanisms, but most studies show an
amelioration of neural plasticity (De Toma et al., in press; Xie et al., 2008).
3.1 Dyrk1A inhibition as one mechanism of action of green tea
extracts containing EGCG
In regards to the treatment of DS cognitive impairment, possibly one of the most interesting property of EGCG is its capability to inhibit the kinase activity of
DYRK1A, a member of the Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family, belonging to the CGMC kinome group. DYRK1A is one of the
major candidate genes to explain DS phenotypes. In DS, the triplication of human
chromosome 21 leads to approximately 1.5-fold higher DYRK1A levels compared
to the general population (Dowjat et al., 2007; Guimerá et al., 1996; Guimera et al.,
1999) and its overproduction has been linked to the neuronal phenotypes and
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cognitive deficits associated with DS (Altafaj et al., 2001). DYRK1A is expressed in
the developing and adult brain and is implicated in cell proliferation and neuronal
development (Dierssen and Martı́nez de Lagrán, 2006; H€ammerle et al., 2003). Its
overexpression leads to disturbances in a wide range of signaling pathways involved
in neural progenitor proliferation and differentiation. This may explain proliferation
deficits (H€ammerle et al., 2011), dendritic branching defects (Martinez de Lagran
et al., 2012; Tejedor and H€ammerle, 2011), or the imbalance of excitation/inhibition
(Souchet et al., 2014) that leads to impaired oscillatory activity in the prefrontal cortex (Ruiz-Mejias et al., 2016). DYRK1A is also involved in neurodegeneration and
neuronal loss appearing in AD (Ferrer et al., 2005) through hyperphosphorylation of
Tau (Ryoo et al., 2007) and APP (Kimura et al., 2007; Ryoo et al., 2008), proteins at
the origin of senile plaques and neurofibrillary tangles.
Given the deleterious effects of DYRK1A overexpression, a plausible therapeutic
strategy for DS would entail controlled inhibition of its expression or the activity
of DYRK1A kinase. Ortiz-Abalia et al. (2008) using inhibitory RNA against
Dyrk1A delivered by bilateral intra-striatal injections of adeno-associated virus
type 2 (AAVshDyrk1A) in 2–3 month-old TgDyrk1A mice, led to a reversal of
corticostriatal-dependent phenotypes, as revealed by the attenuation of their hyperactive behavior, the restoration of motor-coordination defects in the treadmill test,
and an improvement in sensorimotor gating, as detected in the prepulse inhibition
of startle reflex. Using the same strategy in Ts65Dn mice, led to attenuation of
the hippocampal synaptic plasticity defects, accompanied by a normalized thigmotactic behavior in the Morris water maze (MWM), indicating partial improvement of
their hippocampal-dependent search strategy, but with no positive effects in the escape latency or distance traveled (Altafaj et al., 2013). Normalization of the copy
number of Dyrk1A attained by crossing Ts65Dn with heterozygote Dyrk1A +/
mice, resulted in an improvement of the MWM, fear conditioning test and LTP,
but did not rescue behavioral alterations (hyperactivity/attention) nor it improved
the density of mature hippocampal granule cells in the dentate gyrus (Garcı́aCerro et al., 2014). In most studies, normalization of Dyrk1A improves the behavioral
and neural phenotypes of mice overexpressing Dyrk1A but also of Ts65Dn mice.
However, the level rescue is variable depending on the age of the mice, and the
method used for dosage normalization (shRNA or crossings with Dyrk1A +/).
These somewhat positive results obtained encouraged the use of strategies that
could be more translational, as is the use of DYRK1A kinase inhibitors. For this reason, DYRK1A has become a target for DS drug development (Duchon and Herault,
2016) and several molecules have been identified and/or developed to inhibit
DYRK1A activity, including harmine, EGCG, INDY, FINDY, leucettine L41 and
CX-4945 (Adayev et al., 2011; Darwish et al., 2018a,b; Fant et al., 2014; Naert
et al., 2015; Ogawa et al., 2010; Kii et al., 2016; Kim et al., 2016; Stringer et al.,
2017a,b). Harmine, for example, prevents premature neuronal maturation of trisomic
NPCs from the periventricular zone of newborn Ts65Dn mice (Mazur-Kolecka et al.,
2012). These molecules have only recently been developed and have not undergone
extensive preclinical testing and some may have limited pharmacotherapeutic value
3 Dendritic remodelers in Down syndrome
due to side effects [e.g., harmine has side effects associated with monoamine oxidase
A inhibition; (Kim et al., 1997)]. Chronic DYRK1A inhibition using DYR219,
a potent and selective small molecule inhibitor, reduces insoluble forms of amyloid
beta peptides (Aβ) and hyper-phosphorylated tau associated with dramatic delay in
the onset of both amyloid plaques and NFTs (Velazquez et al., 2019). In human
studies, the overexpression of DYRK1A was reported to suppress the activity of
neprilysin (NEP), which is a major Aβ-degrading enzyme in the brain, and phosphorylation at the NEP cytoplasmic domain. NEP activity was markedly reduced in
fibroblasts derived from DS patients compared to healthy controls. This impaired
activity of NEP was rescued by DYRK1A inhibition (Asai et al., 2017).
EGCG is a natural inhibitor of the kinase activity of DYRK1A through noncompetitive inhibition against ATP binding site (Adayev et al., 2006), with an apparent
IC50 of 0.33 μM (Bain et al., 2003; Wang et al., 2012). There are several studies
showing the efficacy of green tea extracts containing EGCG in transgenic mice,
which only overexpress Dyrk1A in an otherwise disomic genetic context. In postweaning (3 week-old) TgDyrk1A mice, 1-month oral treatment with a green tea
extract containing EGCG (2–3 mg/day) induced normalization of the excessive proliferation and accelerated cell cycle exit in the granular cellular layer of the DG
(Pons-Espinal et al., 2013), and the same dose rescued the deficient recognition
memory in 3-month old TgDyrk1A mice as measured with the novel object recognition (NOR) test (De la Torre et al., 2014). These changes were accompanied by a
normalization of hippocampal DYRK1A kinase activity levels, suggesting a potential pharmacological role of EGCG to tackle DS altered neurodevelopment and adult
phenotypes. The same treatment in 3–4 month-old mice mBACtgDyrk1a at a higher
dose (120–200 mg/kg/day EGCG), for 4–6 weeks led to improvement of spine density in prefrontal cortex pyramidal neurons and normalization of LTP (Thomazeau
et al., 2014). In a more recent study in 3–4 month-old mBACTgDyrk1A mice, green
tea extracts containing EGCG at doses of 60 mg/kg/day for 4 weeks rebalanced the
excitation/inhibition since it rescued of glutaminergic expression in the cerebral
cortex and the hippocampus, along with an improvement of spontaneous alternation
(Souchet et al., 2015). Interestingly, when green tea extracts containing EGCG
were given to pregnant mBACTgDyrk1A females, it prevented the alterations in
brain volume and cognitive deficits of their pups (Guedj et al., 2009). Also in the
YACtg152F7 an equivalent dose of 0.6–1 mg/day EGCG using polyphenon 60 a
green tea extract containing EGCG from gestation to adulthood, rescued brain
weight and volume of hypothalamus/thalamus and led to improvement of recognition memory in the NOR test. More recent work has confirmed that classes of
synthetic DYRK1A inhibitors, the leucettines, which cross the blood–brain barrier
and selectively inhibit DYRK1A (Nguyen et al., 2018) leads to normalization of
DYRK1A activity and corrects the novel object cognitive impairment observed in
several DS mouse models (Tg(Dyrk1a), Ts65Dn, Dp1Yey).
Intriguingly, leucettines and green tea extracts containing EGCG only inhibit the
fraction of DYRK1A that is overexpressed, and most native, basal DYRK1A is not
inhibited. This finding is encouraging in terms of potential therapeutic implications,
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as complete inhibition of DYRK1A is not desired. Although the mechanism of such
selectivity in the inhibitory action is not understood, it has been suggested that this
selective effect on DYRK1A overexpression could be linked to the accumulation
of excess DYRK1A and DYRK1A inhibitors in specific cellular compartments
(Nguyen et al., 2018).
Taken together, these results suggested that, in mice, EGCG could partially
rescue some neural and behavioral phenotypes and DYRK1A kinase inhibition is
possibly an important mechanism in the EGCG effects in DS. In fact, if green tea
extracts containing EGCG optimally normalizes DYRK1A activity, additional imbalances of some encoded proteins relevant to DS phenotypic features, such as
APP, SYNJ1 and RCAN could be prevented, as those are phosphorylation substrates
of DYRK1A. Part of those effects could explain the therapeutic effect of green tea
extracts containing EGCG on AD-like phenotypes in DS, also partially mediated by
enhanced tau exon 10 inclusion, leading to an increase in 4R-tau/3R-tau ratio in
differentiated-human neuronal progenitors and in the neonatal rat brains (Smith
et al., 2012). In fact, treatment with EGCG from gestation to adulthood suppressed
3R-tau expression and rescued anxiety and memory deficits in Ts65Dn mouse brains
(Yin et al., 2017). Remarkably, EGCG also potentiates the effects of environmental
enrichment (Catuara-Solarz et al., 2015, 2016).
All these results indicate that it is rather unlikely that the benefits of green tea
extracts containing EGCG in DS are limited to the inhibition of DYRK1A kinase
activity and the beneficial effects observed in Ts65Dn and other partial trisomic mice
are most probably contributed by some of the mechanisms delineated above.
3.2 Effects of green tea extracts containing EGCG on the Ts65Dn
model of Down syndrome
Although certainly important, the fact that green tea extracts containing EGCG
normalized the phenotypes derived from single overexpression of Dyrk1A did not
allow to assume whether it would be also beneficial in an inherently complex trisomic context, with hundreds of over- or under-expressed genes. Already 10 years
ago Xie et al. showed that it was possible to rescue levels of LTP at the Schaffer
collateral-CA1 synapse in hippocampal slices from Ts65Dn mice incubated with
EGCG (10 μM) (Xie et al., 2008). Such rescue of synaptic plasticity suggested a potential benefit of EGCG as pro-cognitive therapy for DS. More recently in NPCs
isolated from the hippocampus of Ts65Dn mice Valenti et al. (2016) showed that
EGCG (20 μM) restored oxidative phosphorylation efficiency and mitochondrial
biogenesis, and improved NPC proliferation, suggesting that EGCG has the potential
to improve neurogenesis alterations in DS, as shown in transgenic TgDyrk1A mice
(Pons-Espinal et al., 2013).
The first study describing the effects of EGCG in vivo in the Ts65Dn model of DS
was performed in adult mice, and showed beneficial effects of green tea extracts
containing 45% EGCG on learning and memory, assessed using the Morris water
maze and novel object recognition tests, in adult (3 months of age) Ts65Dn mice
3 Dendritic remodelers in Down syndrome
(De la Torre et al., 2014). Another study using polyphenon 60 in drinking water at
225 mg/kg/day, containing 27% EGCG (60 mg/kg/day) for 6 weeks in 3–4 monthold trisomic mice confirmed the cognitive amelioration, and have suggested the rescue of the excitation inhibition imbalance as a possible underlying mechanism
(Souchet et al., 2015). In 5–6 month-old mice decaffeinated green tea extracts containing 45% EGCG in drinking water (EGCG dose of 30 mg/kg/day) administered
for 1 month, produced some improvement of the Gallagher index and the thigmotaxis
along learning sessions, suggesting amelioration of the learning strategy, but with
no impact on the latency to reach the escape platform (Catuara-Solarz et al.,
2015). This may suggest that the effects of EGCG can vary with age and that the
dosage range or the bioavailability of EGCG in different preparations may have
an impact of the effects detected.
However, not all the studies provide the same positive results. Two studies by the
same research group, using different doses (high and low; 20 and 50 mg/kg/day) of
EGCG (Sigma Aldrich, >95% purity) showed that neither the short- nor long-term
treatment improved performance in a battery of behavioral tasks and even led to detrimental effects on skeletal phenotypes (Stringer et al., 2015, 2017a,b) even though
previous studies by the same group showed beneficial effects of EGCG on skeletal
phenotypes. Treating Ts65Dn mice at weaning for 3 weeks with the same concentration of EGCG used in human studies (9 mg/kg/day) led to a “substantial
improvement in the postnatal femoral phenotype” at 6 weeks of age, although
it did not significantly lower the DYRK1A activity level, similar to that found by
crossing Ts65Dn with Dyrk1a +/ mice (Blazek et al., 2015). Given that EGCGcontaining supplements are widely available, in a later work, the authors analyzed
six commercially available supplements containing EGCG, and compared two of
those with pure EGCG for their impact on skeletal deficits in a DS mouse model.
The results showed differential effects of commercial supplements on correcting
skeletal abnormalities in Ts65Dn mice, suggesting that the dose of EGCG and
composition of EGCG-containing supplements may be important.
Given the neurodevelopmental load in DS, treatments in the neonatal period
would possibly more efficacious in rescuing brain-related defects of DS. Daily
injection of pure EGCG (25 mg/kg) in postnatal days P3–P15 was found restoration
of neurogenesis, cellularity and connectivity in the hippocampus and neocortex of
Ts65Dn mice, but those effects did not last after treatment cessation (Stagni et al.,
2016). Also, another study in which EGCG was administered to Ts65Dn females
in drinking water (3 mg/day) from mating to weaning of the pups and then, the pups
were fed with EGCG-water, found that EGCG rescued anxiety and memory deficits in
2.5 months Ts65Dn mice and suppressed 3R-tau and promoted 4R-tau expression,
rescuing the dysregulated 4R-tau/3R-tau ratio in Ts65Dn mouse brains (Yin et al.,
2017). A more recent study (Souchet et al., 2019) showed that prenatal treatment with
EGCG-complemented food pellets (50 mg/kg) restored VGAT1/VGLUT1 balance,
and rescued density of GAD67 interneurons in Dp(16)1Yey mice, trisomic for 140
orthologs of chromosome 21, and improved novel object recognition memory, but
not the performance of the Y maze paradigm in the adult (Souchet et al., 2019).
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A very important aspect is that some of the molecular targets of EGCG, such as
DYRK1A, or epigenetic factors, are also modulated by environmental enrichment,
suggesting that EGCG can be considered an “environ-mimetic” drug boosting
activity-dependent plasticity mechanisms. As mentioned above, a recent study
showed that the combination of EE and EGCG acts synergistically in ameliorating
learning alterations and age-related cognitive decline in DS (Catuara-Solarz et al.,
2015), underlining the potential of combinatorial therapeutic approaches.
In summary, the majority of the preclinical studies performed in mouse models
of DS have reported improved behavioral outcomes with EGCG supplements,
which can be variable in the behavioral domains affected and the degree of
improvement. However, there are still many doubts and questions that can only
be verified by well-designed preclinical studies evaluated by multidisciplinary
teams. It is necessary to understand why some studies have shown that EGCG,
even at high doses, does not improve behavioral outcome of Ts65Dn mice and
why it can have even deleterious consequences to bone phenotypes. Several factors
can account for these discrepancies: (i) the composition of the treatment (pure
EGCG vs EGCG in combination with other polyphenols of green tea extracts);
(ii) EGCG dosage and bioavailability; (iii) route of administration (food or drinking water); (iv) duration of the treatment; (v) age of the mice; (vi) species and
strains; (vii) methods used to evaluate cognitive endpoints; (viii) possible effects
of EGCG metabolites or products of its degradation; (ix) the relatively small
sample size in conjunction with the intrinsic phenotypic variability of Ts65Dn
mice. Other possible explanations for the divergent results obtained with EGCG
compounds in adult trisomic mice could also be the different environmental
conditions to which mice had been exposed. Given that Ts65Dn are mainly
subdominant (Martı́nez-Cue et al., 2005), showing less aggressive behavior, one
could think that social defeat or stressful environments such as isolation
could certainly have a deleterious impact. This might impede the effects of EGCG
containing compounds or other therapies to be found in a consistent way, and may
contribute to the lack of effects detected in some studies (Stringer et al., 2015,
2017a,b). Thus, environmental conditions may impact the outcomes and comparability of the studies and would also explain the lack of a clear dosage
dependency, given that the effects are the product of a combinatorial action
of many molecular cascades, and also depend on the basal state, in a kind of
“homeostatic” systems effect. In fact, most of these concerns could be
applied to most preclinical studies not only in DS but also in other intellectual
disabilities.
We certainly need to gain a more systematic view, to understand the variation in
the behavioral and brain neuropathological deviations in DS mouse models, which
will certainly impact the effects of therapy. This can only be achieved by profiling
the effects of the drugs in different mouse strains and DS models in parallel in the
same lab, at different time points and with different doses. Given that DS still has no
therapy to ameliorate intellectual disability, it is certainly worth continuing these
studies.
3 Dendritic remodelers in Down syndrome
3.3 Translational value of preclinical studies with EGCG: Effects in
individuals with Down syndrome
Since EGCG is readily available at reasonable costs, its promising effects in preclinical studies prompted several clinical trials and clinical studies. At the cellular level,
studies in human NPCs and neurons derived from DS-iPSCs treatment with 10 μM
EGCG the same effects detected in mouse cellular models of improved proliferation
and decrease of apoptosis were also detected (Hibaoui et al., 2014). In lymphoblast
and fibroblast cultures from subjects with DS treatment with EGCG (20 μM) rescued
mitochondrial function and promoted mitochondrial biogenesis (Valenti et al.,
2013). This study prompted a case study in a child (10-year and 3-month-old) with
DS who was treated with ECCG (10 mg/kg/day) plus fish oil daily for 6 months
(Vacca and Valenti, 2015). This treatment was safe and improved mitochondrial
function. However, since EGCG was co-administered with fish oil, the extent to
which fish oil contributes to this effect remains to be established.
To the best of our knowledge, only one research group (Hospital del Mar; Spain)
has led the randomized clinical studies using green tea extracts containing EGCG. In
a first pilot double-blind randomized, placebo-controlled phase I clinical trial
(ClinicalTrials.gov ID: NCT01699711, De la Torre et al., 2016), young adults with
DS (29 subjects) aged 14–29 years were treated with either green tea extracts in
capsule form (Mega Green Tea Extract, Decaffeinated, Life Extension®, USA) containing 45% EGCG (mean EGCG oral dose of 9 mg/kg/day; 6 females, 7 males) or
a placebo (8 females, 8 males) for 3 months. The most important aspect of this first
clinical trial was to determine the safety and toxicity of the treatment, since DS
individuals may be more sensitive than euploids to some adverse effects. Even so,
some neuropsychological and quality of life tests were performed after 3 months
of treatment and 3 months after treatment discontinuation. The trial was also
intended to identify DYRK1A activity surrogate biomarkers comparing plasma
homocysteine (hcy) levels (Noll et al., 2009, 2012) at baseline and at the end of
treatment (3 months). Importantly, the biochemical results allowed establishing a
significant correlation between memory improvement and Hcy levels, suggesting
a putative dependence of cognitive improvement on DYRK1A kinase activity
normalization.
An important concern before initiating the clinical trial was hepatic toxicity that
had been shown in several reports, although mainly at higher doses. In the Phase
I clinical study in DS carried out in 2014–2016, no alteration of hepatic function
markers (AST and ALT) was observed. Supporting these observations, in 2018,
the European food safety authority (EFSA) scientific panel on food additives and
nutrient sources added to food (ANS) published a report (Question number:
EFSA-Q-2016-00627; Younes et al., 2018). After reviewing the evidence from 38 intervention studies, which included data on effects of green tea extracts and infusions
on serum transaminases, the panel concluded that exposure to green tea extracts at
doses at or above 800 mg EGCG/day for 4 months or longer, are only associated with
elevations of ALT and AST in a small percentage (usually less than 10%) of the
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population. Moderate or more severe abnormalities in any liver function were observed in 5.1% of the treated subjects in a study with more than 500 subjects treated
with 843 mg EGCG/day for 1 year (Yu et al., 2017). Thus, EFSA experts considered
green tea catechins, including EGCG, to be safe and the rare cases of liver injury that
have been reported after consumption of green tea infusions, to be most probably due
to an idiosyncratic reaction. Animal studies had also reported EGCG to be involved
in redox cycling and quinone formation having both antioxidant and pro-oxidative
activities that may lead to oxidative stress (Dostal et al., 2015; Sang et al., 2005).
However, in the Phase I clinical trial no increase of biomarkers of oxidative stress
(oxLDL and GSH-Px activity) was found, but instead a reduction of lipid oxidation
in subjects treated with EGCG was detected. This improvement of the oxidative/
antioxidative status combined with a healthy lipid profile (Xicota et al., 2019), as
shown by the total cholesterol and LDL cholesterol concentrations, is certainly considered beneficial. This is important since a recent study has carried out an anonymous survey about attitudes and usage of green tea extracts containing EGCG in
individuals with DS completed by caregivers, in which most caregivers who did
not use green tea extracts containing EGCG reported concerns about potential side
effects and lack of effectiveness (Long et al., 2019).
Regarding the cognitive and behavioral effects, after 3 months of treatment,
EGCG-treated individuals showed a significantly higher percentage of correct
answers in visual memory recognition compared with those who had been given
placebo. Three months after treatment discontinuation this effect declined, and treated subjects returned to baseline scores. This indicated that treatment with EGCG
has a positive effect on cognition in DS, albeit moderate and transitory.
In a subsequent clinical study, and based on preclinical studies showing that
green tea extracts containing EGCG potentiated the effects of environmental enrichment (Catuara-Solarz et al., 2016), it was examined whether EGCG could potentiate
the effects of cognitive stimulation, by comparing the effect of cognitive training
with placebo or cognitive training plus green tea extract supplement in capsule form
containing 45% EGCG (Life Extension Decaffeinated Mega Green Tea Extract; Life
Extension ®, USA). The mean EGCG oral dose was the same as in the phase I trial
(9 mg/kg/day). Of course this study does not allow disentangling the effects of
EGCG, but from the therapeutic point of view it was the most promising for the patients. After a power analysis to calculate the sample size, in this double-blind
randomized phase II clinical trial (ClinicalTrials.gov Identifier: NCT01699711), a
larger group (87 subjects) of adults (age: 16–34 years) with DS was enrolled and
treatment lasted 12 months. Including 1 month of treatment with a placebo allowed
to control for the placebo effect, which is very relevant in this population. No placebo
effect was detected in either group (R. De la Torre et al., unpublished). Subjects were
periodically tested with a battery of neuropsychological tests during the 12 months of
treatment, immediately following treatment cessation, and at 6 months after treatment discontinuation. Primary outcome measures were cognitive evaluation, including cognitive domains such as episodic memory, executive function and adaptive
behavior and quality of life. The secondary outcome included neuroimaging in a
3 Dendritic remodelers in Down syndrome
subset of patients measuring regional brain morphology and volume (FLAIR)
sequence, and intrinsic functional organization (i.e., functional connectivity) in
the resting-state within the neural systems, and biochemical biomarkers. Baseline
values of many techniques/variables used in these DS clinical trials had to be validated before evaluating their value for assessing therapeutic effects. For example,
resting-state whole-brain connectivity degree maps generated in 20 DS individuals
and 20 control subjects identified higher regional connectivity in a ventral brain system involving the amygdala/anterior temporal region and the ventral aspect of
both the anterior cingulate and frontal cortices, and lower functional connectivity
in dorsal executive networks involving dorsal prefrontal and anterior cingulate cortices and posterior insula (Pujol et al., 2015). Both correlated with DS scoring on
adaptive behavior related to communication skills.
After 12 months of treatment with EGCG, better performance was detected in
two of the 15 cognitive performance tests and in one of the nine adaptive skills. Specifically, participants treated with EGCG and cognitive training had significantly
higher scores in visual recognition memory (pattern recognition memory test immediate recall, adjusted mean difference: 6.23 percentage points (P ¼ 0.039), inhibitory
control (cats and dogs total score, P ¼ 0.041 and total response time, P ¼ 0.024) and
improved performance of daily tasks requiring basic literacy (ABAS-II functional
academics score, P ¼ 0.002). This last finding was encouraging since benefits in
functional academics imply an improvement in the use of basic literacy skills (reading, writing, and mathematics), allowing a better daily independent functioning (i.e.,
recognize the time, be able to read and write small notes, recognize small quantities
when paying, and performing small operations). Even though these effects were
small and of subclinical magnitude, they were accompanied by a marked enhancement of regional functional connectivity with significant increases in the functional
integration of cortical and subcortical distributed networks, including the frontal
cortex, Wernicke area, the precuneus, occipitotemporal and somatosensory cortices,
and basal ganglia, in the resting-state whole-brain connectivity degree maps (fcMRI)
measurements. Besides, cortical excitability normalization was detected in the noninvasive paired pulse transcranial magnetic stimulation (TMS) studies. These effects
could also contribute to the improved cognitive abilities in individuals treated with
EGCG and cognitive training, since treatment-related changes in functional connectivity within frontal networks were significantly correlated with the increase in
ABAS-II functional academic skills and cognitive deficits in DS have been proposed
to result from an excitation or inhibition imbalance with an excess of synaptic
inhibition in the hippocampus and increased excitation in the cerebral cortex.
It should be remarked that the 2016 study by De la Torre et al., has several limitations: (i) treatment with EGCG was combined with cognitive training and no group
received EGCG only. This was due to the reduced capability of recruiting individuals
willing to participate in a study in which they could be receiving only placebo without
offering any active treatment. Of course this limits the study to determine whether
controlled cognitive stimulation had an effect, and whether EGCG could potentiate
it. Thus, it remains to be established whether EGCG worked by itself in terms of
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improving scores in cognitive measures in individuals with DS or whether it has to be
combined with cognitive training; (ii) In addition, since the green tea extracts contain
various polyphenols in addition to EGCG, the contribution of these polyphenols to
cognitive improvement remains to be clarified and it cannot be ruled out that the
effects ascribed to EGCG were due to interactions with other constituents in the extracts; (iii) the study was not large enough to assess the gender- and/or age-dependent
effects of the treatments. However, gender and age were important co-factor, since
women performed significantly better than men of the same age and IQ in most cognitive tests (De Sola et al., 2015), with the most consistent differences occurring in
memory and executive functioning and negative trends emerged on quality of life
linked to the effect of age after adjusting for IQ and gender, although this was not
frequent. Other important factors may also influence the treatment outcome, such
as genetic variants (Del Hoyo et al., 2016), the progression of neurodegenerative phenotypes in adults (Del Hoyo et al., 2015; Fenoll et al., 2017), quality of sleep (Horne
et al., 2019), self-regulation, psychopathology, daily living/adaptive behavior, and
maladaptive behavior. Finally, factors such as diet, years of education, or thyroid
status, are also important.
Running the trials raised some methodological challenges and questioned the prevailing methodology used to evaluate cognitive functioning of DS individuals.
In fact previous studies had indicated the notable lack of consistency and reproducibility of ratings in some neuropsychological tests used in intellectual disability
(Silverman et al., 2010). This was the motivation to devise and validate the TESDAD
Battery (De Sola et al., 2015) that uses comparison with age-matched typically developed adults. In DS population the TESDAD battery allowed a quantitative assessment of cognitive defects, which indicated language dysfunction and deficits in
executive function, as the most important contributors to other cognitive and adaptive behavior outcomes as predictors of functional change in DS. Concretely, verbal
comprehension and functional academics showed the highest potential as end-point
measures of therapeutic intervention for clinical trials: the former as a cognitive
key target for therapeutic intervention, and the latter as a primary functional outcome
measure of clinical efficacy. Later on, to address this challenge, the National Institutes of Health (NIH) assembled leading clinicians and scientists to review existing
measures and identify those that are appropriate for trials (Esbensen et al., 2017).
The available data in adult individuals with DS confirmed some benefits on
cognitive performance indicating that the preclinical studies had indeed predictive
validity. This is relevant because the validity of the Ts65Dn strain is severly questioned, because of the presence of an extra copy of non-HSA21 genes. However, it is
important to note that it is treatment with EGCG plus cognitive training for
12 months, which is more effective and leads to increased functional connectivity
and normalization of the excitability in the cerebral cortex. Cognitive training
alone had only marginal effects, suggesting that treatments that are ineffective or
scarcely effective if administered alone may elicit some benefits when they are
given in combination with other drug or with cognitive stimulation.
4 Environmental enrichment
4 Environmental enrichment
One of the systems to promote in vivo neuroplasticity in rodents is the use of an
enriched environment (EE) that provides animals an increased physical exercise,
learning experiences, and social interaction. In the context of DS, nowadays, active
care programs are one of the most successful therapeutic interventions used in DS
individuals. Moreover early intervention programs in DS children are the only effective have been developed in an attempt to improve cognitive capabilities. However,
despite resulting in positive results, those are limited and temporary. Environmental
enrichment (EE) in animals mimics a stimulating lifestyle at the laboratory level and
has demonstrated beneficial effects on various aspects of brain structure, function,
behavior and cognition and it leads to functional compensation in different brain
disorders. During developmental stages, it mimics the effects of “early intervention”
involving educational and neuroprotection strategies aimed at enhancing brain
development. A large number of studies have highlighted the fact that EE modifies
the behavior of animals, leading to improvement in complex cognitive functions,
particularly learning and memory, and positively affecting the animal’s emotional
and stress reactivity (Bayne, 2018). The basic experimental setup has hardly changed
over decades: animals, normally rats or mice, are kept in larger groups and in a larger
cage, often equipped with additional toys, nesting material and tubes to hide, and are
compared with what are considered “standard housing” animals in the laboratory,
which in fact could be a paradigm of impoverished environment. EE typically includes: (i) increased physical activity, usually provided by a running wheel and
larger cages, (ii) increased social grouping, and (iii) increased opportunities for
exploration (i.e., larger environments relative to standard housing conditions) and
interactions with novel objects such as toys (Sztainberg and Chen, 2010; Wood
et al., 2011). Some of these components, such as physical activity, per se seem to
provide most of the beneficial effects (Brown et al., 2003; Kobilo et al., 2011;
Lazarov et al., 2010; Van Praag et al., 1999). Voluntary exercise is beneficial for
cognition in both normal rodents and mouse models of altered cognition (Paylor
et al., 1992; Van Praag, 2008; Van Praag et al., 1999). It has been suggested that
these beneficial effects could be mediated, at least in part, by enhanced hippocampal
neurogenesis (Clark et al., 2008).
The first study of EE in a DS mouse model used a paradigm that included an activity wheel, and lasted for 7 weeks after weaning (Martı́nez-Cue et al., 2002). The
study analyzed 86 female and 75 male Ts65Dn mice, and tested circadian spontaneous activity, exploratory behavior, locomotor activity in the open field and spatial
memory in the Morris water maze paradigm (MWM). However, in these experiments
gender was a relevant factor in determining the influence of enrichment on behavior
and learning so that EE improved performance in trisomic females, but deteriorated it
in trisomic males. Such negative influence of EE on Ts65Dn males was interpreted
as derived from the change of the hierarchical organization leading to increased
levels of aggressiveness and stress that could interfere indirectly with their learning
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processes in a rather aversive circumstance, such as the MWM. This was further confirmed in a study using different enrichment conditions, housing in large social
groups or social condition and enrichment in social condition, compared to standard
housing (Martı́nez-Cue et al., 2005). Neither social nor physical stimulation alone
had positive effects, but also did not disturb Ts65Dn mice performance; however,
the combination of both social and physical stimulation deteriorated spatial learning
in trisomic mice that showed subordinate behavior associated to stress, as supported
by the corticosterone levels. Other studies in Ts65Dn mice have shown that voluntary physical exercise improved the cognitive abilities of trisomic mice and enhanced
neurogenesis (Llorens-Martı́n et al., 2010; Parrini et al., 2017).
Exposure of young adult Ts65Dn mice (P60) to EE for 6 weeks induced a marked
recovery of both cognitive and visual functions, reduced brain inhibition and promoted hippocampal synaptic plasticity, but effects in male and female did not differ
(Begenisic et al., 2011). This lack of gender-specific effects may depend on the
differences in the age of onset of EE, but could have also been prevented by using
a protocol of EE in which only 2–3 Ts65Dn males from the same litter were reared
together to avoid an excess of social stimulation that could disturb their behavioral
and learning skills. Using a different paradigm, in which Ts65Dn and control
mothers were breed in enriched or standard conditions, EE resulted in a robust increase in maternal care levels displayed by Ts65Dn mothers (Begenisic et al.,
2015). Maternal care is obviously a very potent stimuli, but living from birth in
enriching conditions also gives the animals the opportunity for enhanced social, cognitive and motor stimulation autonomously experienced by the developing animals
once their reach the weaning age. This EE paradigm led to a normalization of declarative memory abilities and hippocampal plasticity and rescue of visual system
maturation in trisomic offspring. The beneficial EE effects were accompanied by
increased BDNF and correction of overexpression of the GABA vesicular transporter
vGAT (Begenisic et al., 2015).
Moreover, exposure to EE increases hippocampal neurogenesis and the integration of newly born cells into functional circuits. In Ts65Dn mice, both short-term
(P18–P30) and long-term (P18–P45) EE enhanced cell proliferation and neurogenesis in the DG and SVZ both in male and female (Chakrabarti et al., 2011). This result
seems contradictory to the study of Martı́nez-Cue et al. (2002; see above) in which
performance of trisomic males was deteriorated, and was interpreted as differences
in the EE design due to the presence of running wheels. However, in fact both experiments incorporated activity wheels, but given that Chakrabarti et al. did not study
cognitive performance; direct conclusions about the involvement of neurogenesis in
the cognitive effects of EE cannot be extracted.
Rodents living in EE conditions display prominent changes at the anatomical level,
with robust increments in cortical thickness and weight and modifications of neuronal
morphology, in terms of increased dendritic arborization, number of dendritic spines,
synaptic density and postsynaptic thickening, occurring in several regions of the
brain, particularly in the cerebral cortex and hippocampus (Baroncelli et al., 2010;
Hirase and Shinohara, 2014). While 3-week exposure to EE during late development
4 Environmental enrichment
(P30) correlated with 32% higher number of spines in the dendritic arbors of layer III
pyramidal cells of 1-year-old enriched controls, enriched Ts65Dn mice only had 3%
more spines (Dierssen et al., 2003). This suggested that EE does not have long-term
structural plasticity effects in pyramidal cells of the Ts65Dn mouse or, alternatively,
that trisomic mice lose a higher proportion of spines during aging.
At the molecular level, EE in trisomic mice causes a significant restoration of the
G-protein-associated signal transduction systems, which are altered in Ts65Dn mice
(Dierssen et al., 1996) and in DS (Lumbreras et al., 2006) affecting the cholinergic
and noradrenergic systems, which could be beneficial in DS (Dierssen et al., 1997).
EE also significantly increased cyclic AMP production in Ts65Dn mice, while it was
not modified in control animals (Baamonde et al., 2011). Similarly, EE increased
phospholipase C activity in Ts65Dn mice, in response to carbachol and calcium.
DYRK1A is also regulated by EE that normalize DYRK1A levels (Pons-Espinal
et al., 2013).
EE has been also found to either prevent or reverse many neuropathological and
behavioral signs of AD in several transgenic mouse models of this pathology (Sale
et al., 2014; Sansevero and Sale, 2017). In aged Ts65Dn mice, long-term exposure to
EE from P60 until 12 month of age prevents the age-dependent hippocampal increase
of low molecular weight Aβ oligomer isoforms which trigger synapse failure and
memory impairment in AD-like dementia (Sansevero et al., 2016). Aged enriched
Ts65Dn mice, compared to controls reared in standard conditions, showed learning
and memory performances that did not differ from those achieved by euploid
controls (Sansevero et al., 2016).
Interestingly, many of the effects reported for EE, such as neuroplasticity
enhancement, antioxidant activity, anti-inflammatory function, neuroprotection
and promotion of the nonamyloidogenic proteolytic pathway of APP are similar
to those observed upon EGCG treatment. Combined EE-EGCG treatment improved
corticohippocampal-dependent learning and memory. Cognitive improvements were
accompanied by a rescue of CA1 dendritic spine density and a normalization of the
proportion of excitatory and inhibitory synaptic markers in CA1 and dentate gyrus
(Catuara-Solarz et al., 2016). EE has also been shown to normalize the expression
levels and the kinase activity of DYRK1A (Pons-Espinal et al., 2013), and rescued
adult neurogenesis alterations in in mice overexpressing Dyrk1A (TgDyrk1A). However, it is not known whether the same effects are present also in Ts65Dn, although
the levels of APP and MAP2ab are reduced and the levels of SOD1 were increased
upon voluntary exercise, with a slight (10%) reduction of DYRK1A (Kida
et al., 2013).
In spite of the overall positive effect of EE reported in trisomic mice, there are
several contradictory results across labs, and many open questions remain to be
addressed. One confounding factor is that the designs used by laboratories for social,
physical, somatosensory enrichment are generally not uniform. Studies also often
differ in animal sex, and types of controls used (e.g., isolated vs group standardhoused controls) that can further complicate findings and lead to varied outcomes
between laboratories. An important confounder of the social vs nonsocial aspects
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of the enrichment effects is the fact that Ts65Dn males experience higher social
stress when reared in large social groups. It is not surprising then that studies have
given ambiguous and sometimes contradictory results on the efficacy and effectiveness of interventions.
5 Future perspectives
Targeting plasticity impairments in DS has revealed as a promising strategy to
promote cellular mechanisms involved in learning and memory within, and could
lead to improved connectivity among key cognitive brain regions. Promising pharmacological, environmental, and neurostimulation interventions may open a whole
new range of possibilities.
The potential of modulating abnormal plasticity patterns in DS is more promising
during the first years of life when the brain is still under construction. Activitydependent plasticity during the postnatal period helps establishing connectivity
patterns driving the brain maldevelopment. However, it should be taken into account
that plasticity is not equally amenable for therapy in all brain regions and in all lifetime periods. The type of the neural circuit (low-level or high level, single or parallel
processing) dictates the degree of plasticity or stability in response to perturbation
during sensitive or critical periods of development (Harrison et al., 2005).
An interesting possibility is that adult rescue of genetic defects would alleviate or
correct developmental phenotypes by tapping into adult mechanisms of cellular, structural, and behavioral plasticity. In DS, however, the concept of “re-opening” the critical period for neural plasticity has not yet been explored and could be of great interest.
Many preclinical studies have shown that this approach has beneficial effects at the
behavioral, cellular and molecular levels. However, not all have translated into successful clinical trials. Important considerations for the translational value of this preclinical
work are worth to note: (i) when interpreting beneficial preclinical treatment effects, we
should not only consider the statistical significance of the effect but also if the magnitude is sufficient to translate it into a clinically meaningful improvement; and (ii) most
of the studies on DS mouse models have been performed in male mice. In general, all
these aspects may lead to both over- and underestimation of treatment effects and
should be pondered when reading the preclinical studies presented below. Several
guidelines outlining important measures to avoid bias have been prepared including
the ARRIVE guidelines that should be embraced by the DS research community.
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dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A). Arch. Biochem.
Biophys. 507, 212–218.
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Further reading
Guidi, S., Stagni, F., Bianchi, P., Ciani, E., Ragazzi, E., Trazzi, S., Grossi, G., Mangano, C.,
Calzà, L., Bartesaghi, R., 2013. Early pharmacotherapy with fluoxetine rescues dendritic
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induction of hippocampal neurogenesis by long-term environmental enrichment. Ann.
Neurol. 52, 135–143.
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