Amyloid plaque, composed of beta-amyloid (Aβ) is a pathological hallmark of Alzheimer’s disease (AD). Aβ1–42 is a highly neurotoxic and fibrillogenic form of Aβ, and considered as a prime trigger of AD pathogenesis (Glabe 2008). Elevated Aβ1–42 or increase in ratio of Aβ1–42:Aβ1–40 was found in pre-symptomatic familial cases of AD patients (Citron et al. 1992; Cai et al. 1993; Suzuki et al. 1994). Additional copies of the amyloid precursor protein gene cause overproduction of total Aβ, developing early-onset AD (Rovelet-Lecrux et al. 2006). These data indicate Aβ may underlie early neuronal lesions in AD brains.
Sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretase is involved in Aβ. First step for Aβ production is that β-site APP-cleaving enzyme (BACE) 1 cleaves APP to form Aβ N terminus, APPβ and a C-terminal fragment, C99. Next, γ-secretase cut the BACE 1 product to generate Aβs with two variants, Aβ1–40 and Aβ1–42 (Vassar and Citron 2000). In contrast, α-secretase cleaves APP within the Aβ domain to produce APPα and C83, precluding formation of Aβ by competing with BACE1 (Cole and Vassar 2008). Importantly, there is rise in levels of BACE1 and its product (C-terminal fragment of APP) in the sporadic AD brains (Holsinger et al. 2002). Consequently, BACE1 has been considered as a prime therapeutic target for intervention of AD pathogenesis (Fukumoto et al. 2010).
Neuroinflammation has been described as the culprit of AD or, alternatively, as an attempt by the immune system to contain accumulation of Aβ plaques in the brain (Wyss-Coray 2006). Although the role of inflammation in AD is still on debate, accumulating evidence indicates that neuroinflammatory process significantly contributes to pathogenesis of AD (Hwang et al. 2002). The significance of the inflammatory process in AD pathogenesis has been highlighted by epidemiological, retrospective studies reporting a lower incidence of AD in populations receiving long-term treatment with non-steroidal anti-inflammatory drugs (NSAIDs) (Stewart et al. 1997; Walker and Lue 2007; Trepanier and Milgram 2010).
As putative therapeutic tools for AD, various modulators for nuclear factor (NF)-κB signaling pathway have been challenged to modify the neuroinflammatory process in AD models, because NF-κB is a positive regulator for expression of inflammatory molecules including cytokines, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 (Sastre et al. 2006; Kim et al. 2009; Lee et al. 2009a). Anti-inflammatory drug treatment reduces not only neuroinflammatory reactions, but also amyloid deposition in animal models for AD (Yan et al. 2003; Heneka et al. 2005). Significantly, it has been identified that the BACE1 gene promoter region contains NF-κB binding site (Sambamurti et al. 2004). In parallel with the identification, a study demonstrated that BACE1 mRNA and protein levels are increased by proinflammatory mediators and down-regulated by NSAIDs (Sastre et al. 2006).
Magnolia extract contains at least 255 different ingredients such as alkaloids, coumarins, flavonoids, lignans, neolignans, phenylpropanoids and terpenoids (Lee et al. 2011b). Multiple researchers have focused on pharmacological effects of biphenol-structured neolignans including magnolol, honokiol, 4-O-methylhonokiol and obovatol. For instance, compounds isolated from Magnolia family have been shown to own anti-inflammatory (Munroe et al. 2007), neuroprotective (Lin et al. 2006), and antioxidant properties (Fujita and Taira 1994). Furthermore, a potent anxiolytic property of magnolol and honokiol was demonstrated in several studies (Kuribara et al. 1998; Maruyama et al. 1998). Importantly, Ock et al. (2010)demonstrated that obovatol suppressed lipopolysaccharide-induced microglial activation in vitro and in vivo, and exerted neuroprotective effects against neuroinflammation-induced neurotoxicity. We also showed anti-inflammatory properties of obovatol (Kim et al. 2008; Lee et al. 2009b, 2011b).
We employed two different AD animal models including Tg2576 expressing a human APP variant linked to AD and mice that received intracerebroventricular (i.c.v.) inoculation of Aβ1–42, to examine whether obovatol ameliorates cognitive impairments. It has been described that cognitive impairments in both animal models we used in the current study are associated with neuroinflammatory responses (Yan et al. 2001; Kukar et al. 2007; Kotilinek et al. 2008). Therefore, we hypothesize that the natural compound may attenuate Aβ1–42 formation and decline in memory function through blocking NF-κB activity. Here, we show that the compound stabilizes cognitive functions in both animal models for AD.
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- Methods and materials
Alzheimer’s disease is the most common cause of dementia, but etiology of the neurodegenerative disease remains unclear. There is no effective way to cure the disorder or stop the neurodegenerative processes. Thus, much research has been focused on discovering how to intervene AD neurodegeneration. In this study, we examined whether obovatol is able to mitigate memory deficits in AD animal models. This study showed that obovatol improved cognitive function of the animals in the cued and contextual memory tests. Importantly, obovatol ameliorated neuroinflammatory responses and Aβ formation in the cortex and hippocampus of the animal models, and such pharmacological effects might be related to the memory stabilization. Our data also showed that treatment of obovatol suppressed activation of NF-κB, which might contribute to anti-inflammatory and anti-amyloidogenic effects of obovatol, because the transcription factor is a positive regulator for inflammation as well as BACE1 expression (Chen et al. 2011).
Chronic inflammation is associated with a broad spectrum of neurodegenerative diseases including AD (Glass et al. 2010). Increased markers for neuroinflammation such as activated glial cells, proinflammatory cytokines and chemokines are found in or near the pathologic lesions of AD (Wyss-Coray 2006). Furthermore, neuroinflammatory reaction is detected in AD animal models such as Tg2576 and Aβ-infused mice (Benzing et al. 1999; Dong et al. 2011). Critical role of neuroinflammation for AD pathogenesis was highlighted by an epidemiological study where NSAIDs reduce AD incidence by an average of 58% (Szekely et al. 2004). NSAIDs also have been shown to reduce levels of highly amyloidogenic Aβ1–42 peptide and Aβ deposition in a mouse model of AD (Lim et al. 2000). In agreement with the notions, obovatol treatment significantly attenuated neuroinflammation and this effect was accompanied by the improved cognitive performance. Thus, anti-inflammatory compounds including obovatol might be potential therapeutic tools for AD neurodegeneration.
There was rise in escape distance and escape latency 24 and 48 h after Aβ1–42 peptide infusion. Somebody may argue that the impairments come from sensorimotor deficit caused by acute effects of surgery. However, we did not observe any sensorimotor deficits in the animals during the tests. Furthermore, Aβ1–42-infused animals exhibited impairments in spatial memory as determined by probe test (Fig. 3). We assume that the Aβ1–42-induced increase in escape latency is related to neuroinflammation. Neuroinflammatory reactivity was shown by immunostaining for GFAP and increased DNA-binding activity of NF-κB (Fig. 4). Ameliorated neuroinflammation by obovatol treatment was associated with restoration of cognitive performance. In supportive to this study, multiple other studies demonstrated positive correlation between Aβ-mediated neuroinflammation and memory deficit (Craft et al. 2006; Ralay Ranaivo et al. 2006; Medeiros et al. 2010). It is not clear which inflammatory pathway is responsible for the deterioration as of now, but Medeiros et al. demonstrated that TNF-alpha signalling is related to cognitive decline induced by Aβ. Thus, further study is required to elucidate which specific pathway causes Aβ-induced cognitive impairments.
A variety of Aβ aggregates including dimers, soluble oligomers, protofibrils, diffuse plaques, and fibrillar deposits can be seen in the senile plaques (Caughey and Lansbury 2003). It is believed that the fibrillar form of Aβ peptide is generated in the early stages of AD (Drouet et al. 2000), which causes neuronal dysfunction and death (Yankner 1996). In support of the suggestion, neurotoxicity of Aβ peptide has been demonstrated (Emre et al. 1992). In this study, we showed that i.c.v. infusion of Aβ1–42 fibril causes memory impairments. Moreover, we detected that treatment of Tg2576 mice with obovatol significantly lowered cognitive deterioration and this effect was coincided with 35% and 25% reduction in Aβ1–42 concentration in the hippocampus and cortex, respectively. Aβ1–42 is derived from APP by sequential actions of BACE1 and γ-secretase. BACE1 is essential for initiation of Aβ1–42 generation and the enzyme expression is up-regulated in AD brains (Holsinger et al. 2002; Sastre et al. 2006; Cole and Vassar 2008). It is likely that BACE1 expression is regulated by neuroinflammatory events (Sastre et al. 2008). Importantly, NF-κB controls expression of BACE1, and activation of the inflammatory transcription factor enhances Aβ formation (Sambamurti et al. 2004; Chen et al. 2011). Therefore, therapeutic tools targeting NF-κB would likely be of benefit in the treatment of AD (Sastre et al. 2006). There is abundant evidence that NF-κB activity is associated with amyloidogenesis. For instance, (−)-epigallocatechin-3-gallate, a compound from green tea rescues cognitive function and reduces β-secretase activity through inhibition of NF-κB pathway in preseniline 2 mutant mice (Lee et al. 2009a). Further support comes from a report that the mutation on the NF-κB binding site of BACE1 promoter decreases the promoter activity resulting in reduced expression of BACE1 (Bourne et al. 2007). In this investigation, we observed that obovatol suppressed DNA-binding activity of NF-κB and Aβ contents in the brains of Tg2576 mice (Figs 6 and 9), suggesting that obovatol may reduce amyloidogenesis by way of blunting NF-κB signaling pathway.
This study showed that obovatol (0.5–50 μg/mL) can inhibit Aβ aggregation or promote its destabilization. Moreover, electron microscopy scanning also strongly supports this notion, as suppression of Aβ fibril generation by obovatol was directly visualized. We and others have shown that natural compounds such as (−)-epigallocatechin-3-gallate and polyphenols had an anti-fibrillogenic property (Ehrnhoefer et al. 2008; Riviere et al. 2008; Lee et al. 2009a). Obovatol seems to have a comparable inhibitory effect with such reported compounds. It has been described that memory deficits in middle-aged Tg2576 mice are caused by extracellular deposition of 56 kDa soluble Aβ assembly (Lesne et al. 2006). Oral intake of obovatol reduced memory impairments of Tg2576 mice. Thus, it can be assumed that attenuation of Aβ production and aggregation by obovatol rescued from cognitive deterioration in Tg2576 mice.
We observed robust ameliorative effects of obovatol in the passive avoidance tests (Fig. 5d). Anti-inflammatory property of obovatol might contribute to the results at least in part. However, it is possible that potential effects of obovatol on anxiety-like behavior have contributed to the observed cognitive performance. Compounds isolated from Magnolia extract such as 4-O-methylhonokiol, honokiol and magnolol were shown to have anxiolytic properties (Kuribara et al. 1998; Maruyama et al. 1998; Han et al. 2011). Interestingly, obovatol was also demonstrated to possess anxiolytic properties (Seo et al. 2007). We could not find any studies that Magnolia extract or obovatol increases anxiety-like behavior. But we cannot rule out the possibility that potential effects of obovatol treatment on anxiety-like behavior contribute to their delayed entrance into the dark.
Magnolol, honokiol and obovatol are major compounds contained in Magnolia officinalis (Shen et al. 2009). They share neolignan structures, and are assumed to have similar pharmacokinetic behaviors. Without pharmacokinetic data, it is hard to predict whether obovatol reaches to the brain to exert its pharmacological effects, but it is noteworthy that concentration of magnolol is fourfold higher in the mouse brain than in plasma when the compound (5 mg/kg) is injected intravenously, indicating that magnolol could pass through the blood–brain barrier, and act on the brain (Tsai et al. 1996). Similar dose of obovatol to that in current study elicits anti-neuroinflammatory (10 mg/kg × 4 days) and anxiolytic effects (0.2, 0.5 and 1.0 mg/kg), supporting that obovatol is able to reach to the brain and act on the central nervous system (Ma et al. 2009; Ock et al. 2010). In the previous study, we have shown that extract of Magnolia officinalis (5 and 10 mg/kg for 1 week) protects against scopolamine-induced memory impairments (Lee et al. 2009c). It is assumed that the extract contains about 0.1–0.3% of obovatol (Lee et al. 2011b). Based on the data, we tried 0.03 mg/kg dose of obovatol in preliminary studies. However, we did not detect significant protective effect on memory deficits in animal models. Thus, we increased the dose up to 1 mg/kg for further studies. It was described that magnolia bark extract did not cause any toxic effects in rats treated with two different doses of 480 mg/kg for 21 days and 240 mg/kg for 90 days (Liu et al. 2007). Furthermore, the oral LD50 for the extract is higher than 50 g/kg in mice, and treatment mice with magnolia extract (2500 mg/kg) did not cause any clinical signs of toxicity or mortality (Li et al. 2007). These data indicate that obovatol could be safe and effective to apply to clinical studies.
Overall, these results showed that obovatol isolated from Magnolia officinalis unequivocally attenuates AD-like abnormalities including cognitive impairments and neuroinflammatory reactions in AD animal models suggesting this compound could be eligible for intervening neuronal dysfunction in AD brains.