3.6 LSL60101 effects on astroglial activation and synaptic
dysfunction.
All treatments were able to attenuate astrogliosis in the hippocampus of
5XFAD brains by decreasing GFAP immunoreactivity in DG, CA1 and CA3
areas in 5XFAD mice groups in comparison with untreated mice (Figures
6a-d). Likewise, synaptic plasticity markers were evaluated by WB.
Decreases in the protein levels of postsynaptic density protein 95
(PSD95) and synaptophysin (SYN) were determined in 5XFAD mice when
compared to WT mice (Figures 6f-6h). I2-IR ligand LSL60101 increased
PSD95 levels when compared to 5XFAD control (Figure 6f, 6h). SYN levels
were found increased in LSL60101, and donepezil/LSL60101 treated 5XFAD
mice, reaching significance only for the combination-treated group.
Donepezil treatment was not able to significantly modify these markers
(Figure 6g, 6h).
DISCUSSION
The identification of new targets for AD treatment is required due to
the lack of effective disease treatment. At present, AChEI are one of
the standard therapeutic options clinically available for AD patients;
however, those treatments provide only symptomatic benefit in AD
(Sinforiani et al., 2003; Rosini et al., 2014). Fortunately, the number
of disease-modifying drugs targeting AD hallmarks such as aducanumab
(BIIB037), which is currently in phase 3 trials, is increasing (Cummings
et al., 2020). Combination therapies of symptomatic and
disease-modifying drugs have centred attention due to the multifactorial
origin of the disease, and most current clinical trials combine
donepezil with novel neuroprotective drugs (Frölich et al., 2019).
However, it remains a challenge that must be addressed to unveil new
strategies that could be more effective in disease-modifying treatment,
rather than address symptoms (Schmitt et al., 2004).
Several studies have described the symptomatic effects of donepezil in
animal models of dementia and AD, but few in vivo studies have
evaluated donepezil neuroprotective effects regarding the
disease-modifying actions of this compound alone or in combination
(Jiangbo and Liyun, 2018; Krishna et al., 2020; Yang et al., 2020;
Ongnok et al., 2021). Here, we studied the effect of chronic low doses
of an I2IR ligand, donepezil and their combination.
In the light of our studies, we demonstrated for the first time the
neuroprotective effects of selective I2-IR ligands in the
senescence-accelerated mouse prone 8 (SAMP8), a mouse model of
late-onset AD (Griñán-Ferré et al., 2019). LSL60101, a selective I2-IR
ligand has been shown to induce several biological effects associated
with I2-IR occupancy and most importantly neuroprotective effects in the
CNS (Menargues et al 1994; Boronat et al., 1998; Casanovas et al., 2000;
Sánchez-Blázquez et al., 2000).Therefore, it represents a suitable drug
candidate to validate this receptor as a target for AD. Here, we
demonstrated the efficacy of chronic low-dose I2-IR ligand LSL60101
treatment in comparison with donepezil by assessing the beneficial
outcomes in a model of familial AD.
Cognitive abilities are the essential indicators to unveil
pharmacological effects in AD. Firstly, chronic low-dose treatment with
the I2-IR ligand LSL60101 or donepezil reversed the cognitive deficits
presented by 7-month-old 5XFAD mice, without affecting WT mice in the
NORT paradigm. However, in the spatial memory test, only LSL60101 showed
improvements in memory. Likewise, 5XFAD exhibited improved social
behaviour after LSL60101 or donepezil treatment. In agreement with these
results, donepezil has been shown to improve social interactions in
scopolamine-induced memory impairments in mice (Riedel et al., 2009) and
in drug-trials for AD (Boada-Rovira et al., 2004). Nevertheless, the
beneficial effect of an I2-IR ligand treatment on social interaction
deficits has not been described previously.
By contrast, I2-IR ligand LSL60101 did not modify anxiety-like
behaviour, albeit previous studies have shown the in vivoanxiolytic and anti-depressant-like effects induced by I2-IR ligands
(Finn et al., 2003; Tonello et al., 2012). Interestingly, the absence of
anti-depressant effect after treatment with LSL60101 in healthy rats was
recently described (Hernández-Hernández et al., 2020) further supporting
our results, since anxiety-like and depressive-like behaviour are
strongly associated, sharing common molecular pathways (Gatt et al.,
2009). In contrast, and according to literature (Fitzgerald et al.,
2020), chronic treatment with donepezil showed beneficial effects on the
anxiety-related disturbances exhibited by 5XFAD mice .
Recently, we reported that the amyloidogenic APP processing pathway was
suppressed in SAMP8 and 5XFAD mice after treatment with novel I2-IR
ligands, anticipating the role of I2-IR modulation in the Aβ biogenesis
(Griñán-Ferré et al., 2019; Abás et al., 2020; Vasilopoulou et al.,
2020b). Accordingly, in this study, we also demonstrated that chronic
low-dose treatment with I2-IR ligand LSL60101 attenuated the amyloid
plaque burden in 5XFAD mice. In addition, Aβ plaques reduction was
accompanied by a decrease in CTFs and Aβ hippocampal protein levels, as
well as favourable modifications in APP processing after treatment.
Conversely, recently it was reported that the I2-IR ligand BU224 does
not ameliorate Aβ amyloidosis in 5XFAD mice (Mirzaei et al., 2020), but
improves memory. In contrast with LSL60101 like molecules, BU224 blocked
the memory-enhancing effect of agmatine in memory deficits induced by
Aβ1-42 in mice (Kotagale et al., 2020). These
discrepancies between I2-IR ligands can be explained by differences in
compound administration conditions such as dose, time (sub-chronicvs. chronic) and route of administration. Thus, we hypothesise
that low doses of LSL60101, as well as the chronicity of treatment, has
a clear beneficial effect on amyloid burden because of differential
characteristics among I2-IR ligands (Sánchez-Blázquez et al., 2000;
Garau et al., 2013).
Several studies have demonstrated the effect of donepezil on Aβ
pathology in AD models, reporting either beneficial changes (Dong et
al., 2009; Takada-Takatori et al., 2019) or lack of effect (Ju and Tam,
2020). Here, low-dose donepezil treatment did not induce significant
changes on neither Aβ plaques nor APP processing in 7-month-old 5XFAD.
Of note, co-administration of I2-IR ligand LSL60101 with donepezil was
shown to have a greater effect on APP processing, as the co-treatment
donepezil/LSL60101 induced an increase in Aβ degradation enzymes gene
expression in 5XFAD mice, which was not determined in the other treated
groups. To sum up, this is the first time that an I2-IR ligand was shown
to be effective in reducing Aβ burden in in vivo mice model of
AD.
Presence of p-Tau, another major AD hallmark, in the 5XFAD model is
supported by previous studies suggesting that Tau pathology may be
downstream from Aβ pathology (Blanchard et al., 2003; Saul et al.,
2013). I2-IR ligand LSL60101 ameliorated Tau pathology in the
hippocampus of 5XFAD mice. Interestingly, it was shown recently that
chronic treatment with idazoxan, a mixed α2/I2 ligand, reduced p-Tau
reversing cognitive deficits in AD mice, because of its α2 blockade
action (Zhang et al., 2020). In this case, the effect of LSL60101 on Tau
pathology can be attributed to its I2 selectivity, more than to the α2
one. Surprisingly, the p-Tau reduction reached significance in the 5XFAD
mice treated with the combination of LSL60101 with donepezil
demonstrating, in this case, a putative additive effect of the drugs on
Tau pathology. Indeed, amelioration of Tau pathology has been induced in
AD animal models both by donepezil (Yoshiyama et al., 2010) and by I2-IR
ligand treatments (Griñán-Ferré et al., 2019; Vasilopoulou et al.,
2020b). It is possible that the activation of distinct molecular
pathways by the two molecules with different modes of actions resulted
in a remarkable p-Tau reduction observed in the donepezil/LSL60101
treated mice group.
It is well-established that Aβ accumulation jointly with p-Tau increases
microglial activation and inflammatory mediators’ production in AD
brains (Akiyama et al., 2000; Serrano-Pozo et al., 2011; Zhang and
Jiang, 2015). On the one hand, chronic low-dose LSL60101 treatment
reduced microgliosis in 5XFAD mice in contrast to the standard of care
donepezil, explaining the decrease in the amyloid deposition that in
turn would lead to a decrease in gliotic response after LSL60101
treatment. On the other hand, inflammatory gene expression increase
(Il-1β, Il-6, and Ccl12 ) was observed after treatment with
I2-IR ligand LSL60101 but not with donepezil. Interestingly, this is
further supported by a significant upregulation of Trem2 gene
expression determined in the LSL60101 treated mice, further confirming
the neuroinflammatory modulation by I2-IR ligand LSL60101 (Hwang et al.,
2010; Griñán-Ferré et al., 2019; Vasilopoulou et al., 2020a).
Furthermore, increased Trem2 expression has been shown to
reprogram microglia responsivity mediating microglial cytokine release,
migration and clearance of Aβ deposits, ameliorating neuropathological
and behavioural deficits of AD mouse models (Lee et al., 2018; Zhao et
al., 2018).
It has been described that the I2-IR modulate the expression of
astrocyte marker GFAP, especially considering their primary location in
astrocytes (Regunathan et al., 1993; Olmos et al., 1994). GFAP
diminution was observed in SAMP8 after chronic treatment with selective
I2-IR ligands (Griñán-Ferré et al., 2019; Vasilopoulou et al., 2020b).
In agreement with those results, chronic low-dose treatment with I2-IR
ligand LSL60101 attenuated astrogliosis in 5XFAD mice. By contrast, it
has been shown that chronic treatment with LSL60101 increased GFAP
immunoreactivity (Alemany et al., 1995) resulting in reactive
astrocytosis and preventing motoneuron cell death in neonatal rats
(Casanovas et al., 2000). However, here, in a neurodegenerative
landscape provided by the 5XFAD model, the diminution of GFAP reactivity
ran in parallel with the attenuation of the Aβ pathology and microglial
activation observed after LSL60101 and donepezil treatment, given
further support to the beneficial effects of I2-IR ligand on mice
behaviour. Ultimately, we demonstrated that chronic low-dose treatment
with I2-IR ligand as well as donepezil enhanced synaptic plasticity,
further supporting the cognitive and behavioural improvement induced by
the LSL60101 in 5XFAD mice.
CONCLUSIONS
Collectively, we report that chronic low-dose treatment with I2-IR
ligand LSL60101 reversed cognitive deficits in 5XFAD mice, changing AD
neuropathological hallmarks, including glial activation and synaptic
dysfunction. Strikingly, treatment with I2-IR ligand LSL60101 was found
to exert more significant beneficial effects under neurodegenerative
process caused by Aβ pathology than donepezil, However, combination
treatment only showed discrete synergistic effects at the molecular
level (e.g., tau hyperphosphorylation or synaptic plasticity),
suggesting that increased dosage and/or duration of the treatment may be
able to produce better effects on both behaviour and AD-hallmarks,
targeting simultaneously pathological and symptomatic reliefs. In
conclusion, our findings demonstrate the therapeutic potential of the
I2-IR for AD treatment as a disease-modifying single therapy and provide
new insights for their efficacy.