In the present study, we documented that Z-GS exerted protective effects on cognitive and neuropathological impairments in the transgenic mouse models of AD, which was manifested in improved cognitive defects, decreased Aβ production and deposition, alleviated inflammatory response and glial activation, and reduced synaptic loss. The mechanism is probably attributed to down-regulation of BACE1 expression through inhibiting the TLR4/NF-κB signaling pathway.
The detrimental role of chronic inflammation together with the neurodegenerative events involved in AD has been widely explored for new potential therapeutic approaches. Due to the known contribution of innate immune and inflammatory response, TLR4 has received increased attention and thus representing a promising target for inflammation-based AD therapy [23]. A number of studies have shown that the expression of TLR4 is abnormally increased in the brains of AD patients and mice with AD-like pathology [24–26]. High TLR4 immunoreactivity was observed in glial cells surrounding Aβ plaques in postmortem brains of AD patients [24], and significantly higher levels of pro-inflammatory cytokines have been detected in the brains of APP/PS1 mice [27]. Moreover, oligomeric and fibrillar Aβ peptide promoted the discharge of pro-inflammatory and neurotoxic cytokines IL-1β, IL-6, and TNF-α by microglia and astrocytes in a TLR4-dependent manner [28]. Notably, intracerebroventricular injection of Aβ induced an inflammatory response leading to neuronal death, synaptic loss, and cognitive impairment in wild-type mice but not in TLR4 knockout mice [10]. However, TLR4 antagonists were found to abolish Aβ oligomer-induced astrocyte activation, neuronal cell death, and long-term potentiation deficit in hippocampal slices [29]. Collectively, these findings suggest that specific inhibition of TLR4 is likely a potential therapeutic strategy for AD. In accordance with the previous studies [26, 30], we found that TLR4 expression levels were significantly increased in the brains of APPswe/PS1dE9 mice, indicating TLR4 signaling pathway was excessively activated in the brains of mice with AD-like pathology. Nevertheless, chronic administration of Z-GS in the APPswe/PS1dE9 mice strongly inhibited TLR4 expression down to the basal levels of wild-type mice. Furthermore, Z-GS dramaticaly inhibited the over-phosphorylation of IκBα and p65 and markedly attenuated inflammatory response by decreasing pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the brains of APPswe/PS1dE9 mice. Taken together, these results demonstrate that the neuroprotective role of Z-GS against chronic inflammation in APPswe/PS1dE9 mice is probably attributed to its anti-inflammatory capability by suppressing the TLR4/ NF-κB pathway.
Many lines of evidence support that Aβ peptides and amyloid plaques play important roles in the pathogenesis of AD [31]. BACE1 is required for the generation of Aβ, which aggregates into bioactive conformational species and likely initiates toxicity in AD. Given the increased concentration and activity of BACE1 in AD brain, it is thereby making BACE1 a prime target for slowing down Aβ production in AD [32, 33]. In the present study, Z-GS treatment were found to markedly reduce the production and deposition of Aβ in the APPswe/PS1dE9 mice relative to the transgenic controls, showing that Aβ-initiated pathological cascades in AD might be restrained by Z-GS treatment. To further elucidate the mechanisms responsible for Z-GS-modulated decrease in Aβ generation and accumulation, we measured the levels of full-length APP, the major secretases (ADAM10, BACE1, and PS1) involved in APP metabolism, and enzymes for Aβ degradation (NEP and IDE). We documented that Z-GS treatment significantly depressed the expression of BACE1 and the release of its cleavage product sAPPβ in the brains of APPswe/PS1dE9 mice, suggesting that Z-GS treatment was likely to suppress amyloidogenic APP pathway mediated by BACE1 in AD brain. By contrast, the expression levels of full-length APP, PS1, ADAM10 and its cleavage product sAPPα as well as NEP and IDE were not affected by Z-GS treatment in the APPswe/PS1dE9 mice, indicating that the Aβ decease was not regulated by the synthesis and α-cleavage proteolytic processing of APP and degradation of Aβ. Although there is no evidence that TLR4 directly regulates BACE1 expression, NF-κB signaling has been shown to enhance BACE1 expression in the brain [34, 35]. Considering the activation of NF-κB signaling is a downstream event provoked by TLR4, it appears that the Aβ reduction mediated by BACE1 with Z-GS treatment in the brains of APPswe/PS1dE9 mice may be attributed to its selective inhibition of TLR4/NF-κB pathway.
There has been abundant research in recent years to explain the role of gliosis in the development of AD [36]. It has been demonstrated that Aβ plaque formation and reactive gliosis are required for induction of cognitive deficits in mouse models of AD [37]. Moreover, presence of activated glias and inflammation-related mediators association with the Aβ lesions was shown in the brains of patients with AD pathology [38]. Compared with the transgenic controls, we found that Z-GS treatment remarkably alleviated the hyperplasia of astrocytes and microglia in the APPswe/PS1dE9 mice in the present study. A number of studies have implied that Aβ peptides induce the proliferation and activation of astrocytes and microglia [39, 40]. It seems that the dramatical decrease of reactive gliosis observed in the brains of APPswe/PS1dE9 mice treated with Z-GS might be secondary to the reduction of Aβ assemblage. However, there is increasing evidence showing that TLR4/NF-κB signaling plays a crucial role in astrocyte and microglial activation [41, 42]. Therefore, the significant reduction of activated astrocytes and microglia in the Z-GS-treated APPswe/PS1dE9 mice may result from the inhibition of TLR4/NF-κB pathway rather than the decline of Aβ accumalation.
Synapse loss and synaptic dysfunction are critical processes involved in the pathophysiology of AD, which are more directly tied to the severity of cognitive defects [43]. Compelling evidence suggests that soluble Aβ peptide oligomers induce synaptic loss in AD [44]. Moreover, it has been reported that hippocampal accumulation of Aβ is responsible for reducing dendritic protein MAP2 and dendritic spines and hippocampal based learning and memory impairments [45]. In addition, IL-1β and TNF-α were found to suppress hippocampal long-term potentiation directly at the synapse [46]. Accordingly, the mechanisms underlying the restoration of impaired synaptic plasticity and integrity in the Z-GS-treated mouse models of AD in the present study is probably attributed to its multiple effects in reducing Aβ production and neuroinflammation.
Cognitive impairment is the major clinical hallmark of AD and gradually worsens as the disease progresses [47]. There is an urgent need to develop therapeutic strategies that may alleviate or reverse the symptoms of cognitive deficits in AD. Consistent with our previous studies [19, 20], the 12 months old APPswe/PS1dE9 mice treated with vehicle showed marked spatial learning and memory impairments compared with wild-type mice. Nevertheless, TLR4 inhibition with Z-GS treatment significantly improved the cognitive defects in the mouse models with AD-like pathology. Notably, a number of studies have exhibited that suppression of TLR4 activation attenuates Aβ or lipopolysaccharide-induced cognitvie impairment [48, 49]. In accordance with these findings, we indicated that Z-GS treatment dramatically inhibited TLR4 activation, contributing to Aβ reduction, neuroinflammtion alleviation, and synaptic dysfunction recovery in the brains of APPswe/PS1dE9 mice. Therefore, the beneficial effect of TLR4 inhibition with Z-GS treatment on cognitive improvement in the mouse models of AD is likely result from the amelioration of diverse neuropathological damages.