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.