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- Material and methods
Alzheimer’s disease (AD) is characterized by senile plaques and neurodegeneration although the neurotoxic mechanisms have not been completely elucidated. It is clear that both oxidative stress and inflammation play an important role in the illness. The compound curcumin, with a broad spectrum of anti-oxidant, anti-inflammatory, and anti-fibrilogenic activities may represent a promising approach for preventing or treating AD. Curcumin is a small fluorescent compound that binds to amyloid deposits. In the present work we used in vivo multiphoton microscopy (MPM) to demonstrate that curcumin crosses the blood–brain barrier and labels senile plaques and cerebrovascular amyloid angiopathy (CAA) in APPswe/PS1dE9 mice. Moreover, systemic treatment of mice with curcumin for 7 days clears and reduces existing plaques, as monitored with longitudinal imaging, suggesting a potent disaggregation effect. Curcumin also led to a limited, but significant reversal of structural changes in dystrophic dendrites, including abnormal curvature and dystrophy size. Together, these data suggest that curcumin reverses existing amyloid pathology and associated neurotoxicity in a mouse model of AD. This approach could lead to more effective clinical therapies for the prevention of oxidative stress, inflammation and neurotoxicity associated with AD.
Alzheimer’s disease (AD), characterized by progressive memory loss, cognitive deterioration and behavioral disorders is the most common cause of dementia among elderly people. AD is diagnosed in postmortem analysis by the presence of neurofibrillary tangles, senile plaques, and neuronal loss. The accumulation of amyloid-β (Aβ) aggregates as soluble oligomers, ADDLs, and senile plaques plays a key role in the pathogenesis of AD (Selkoe 1994). There is also increasing evidence supporting the role of cerebrovascular amyloid angiopathy (CAA) as a contributing factor to dementia (Jellinger 2002; O’Brien et al. 2003). Senile plaques have been associated with synaptic loss and abnormal neuritic morphology (D’Amore et al. 2003; Lombardo et al. 2003; Brendza et al. 2005) leading to a disruption of cortical synaptic integration (Stern et al. 2004). Although the ultimate neurotoxic mechanisms have not been completely elucidated, it is well-established that the altered microenvironment around plaques is responsible, at least in part, for the pathological neurites observed in AD (Hashimoto and Masliah 2003). Roles for inflammation and oxidative damage have also been implicated in neurodegeneration, and may play an important role in AD (Christen 2000; Cole et al. 2004). Aβ can produce H2O2 (Huang et al. 1999) and free radicals associated with plaques may mediate plaque-induced toxicity (El Khoury et al. 1998; McLellan et al. 2003; Garcia-Alloza et al. 2006a).
The natural product curcumin acts through a spectrum of activities and represents a hopeful approach for delaying or preventing the progression of AD (Cole et al. 2004). Curcumin is a yellow pigment extracted from the rhizome of the plant Curcuma longa (Bala et al. 2006) and in vitro studies have shown that curcumin attenuates inflammatory response of brain microglial cells (Kim et al. 2003; Jung et al. 2006). Curcumin also inhibits the formation of Aβ oligomers and fibrils in vitro (Ono et al. 2004; Yang et al. 2005). Other studies have shown that curcumin prevents neuronal damage (Shukla et al. 2003), and reduces both oxidative damage (Lim et al. 2001) and amyloid accumulation (Yang et al. 2005) in a transgenic mouse model of AD. Clinical trials with curcumin have shown that the compound is not only safe but may be a chemoprotective (Cheng et al. 2001) and anti-inflammatory (Holt et al. 2005) drug. In the present work, we used multiphoton microscopy (MPM) and longitudinal imaging to evaluate in vivo and in real time the effect of systemic curcumin administration on existing Aβ deposits using aged APPswe/PS1dE9 transgenic mice. We also assessed the effect of curcumin on the dendritic abnormalities associated with dense-core plaques. We found that curcumin clears and reduces plaques, and partially restores the altered neurite structure near and away from plaques, adding evidence that curcumin has beneficial effects in reducing the pathology and neurotoxicity of AD in transgenic mice.
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- Material and methods
Alzheimer’s disease is characterized by several pathological features that include senile plaques. Plaques contain a myriad of molecules, apart from Aβ, including apolipoprotein E, phosphorylated tau and many others (Armstrong 2006). Although the exact role of these various bioactive compounds is difficult to dissect, the microenvironment surrounding plaques is a local source of oxidative stress (Behl and Moosmann 2002). Plaque deposition is tightly associated with neurotoxicity, as exhibited by dystrophies and distorted neurites (Knowles et al. 1999; Le et al. 2001; Garcia-Alloza et al. 2006a). However, soluble aggregates of Aβ including oligomers and ADDLs may contribute to neurotoxicity (Selkoe 1994).
In the present study, we show that curcumin stains amyloid deposits ex vivo, confirming previous studies in other transgenic mouse models and in tissue sections from AD patients (Yang et al. 2005). When we assessed the capacity of curcumin to cross the blood–brain barrier and label amyloid deposits in vivo, we observed using MPM that after a single i.v. dose the staining with curcumin was undetectable, despite intravascular fluorescence. However, after administering curcumin i.v. daily for 7 days we observed that senile plaques and CAA were stained with curcumin, demonstrating that it crosses the blood–brain barrier and, importantly, accumulates in the local vicinity of senile plaques. This observation supports a trial where radiolabeled curcumin was evaluated as a ligand for non-invasive amyloid imaging (Ryu et al. 2006). The dose of curcumin that we used (7.5 mg/kg/day) is less than the ‘low dose’ (24 mg/kg formulated in chow) of previous reports (Lim et al. 2001; Yang et al. 2005), and is much lower than tolerable doses approaching 2000 mg/kg (Lim et al. 2001). While careful toxicological testing will be needed, the dose used in this study resulted in no observable side effects in the mice.
When we examined the effect of curcumin treatment in vivo on amyloid deposition we observed that curcumin was capable of reducing plaque burden. This occurred by preventing formation of new deposits, clearing existing deposits, and reducing the size of remaining deposits. Together, these data confirm that curcumin can not only suppress new amyloid accumulation, as observed in tissue sections from chronically treated animals (Yang et al. 2005), but it can also remove the previously deposited amyloid. This can be attributed to the capacity of curcumin to disaggregate and inhibit Aβ aggregation as previously described in vitro (Ono et al. 2004; Yang et al. 2005). However, due to the broad range of activities of curcumin, it cannot be excluded that some other actions are contributing to the observed clearance of amyloid. In this sense, other well-known anti-oxidants such us Ginkgo biloba extract (EGb 761) or vitamin E do not seem to affect Aβ deposition or clearance (Garcia-Alloza et al. 2006a) and non-steroidal anti-inflammatory drugs such as ibuprofen show a surprisingly limited effect on inflammatory markers in APPswe transgenic mice when compared with curcumin (Cole et al. 2004). However, no study has systematically assessed a broad range of anti-oxidants and anti-inflammatory drugs in the same paradigm.
With regard to biochemical measures of Aβ, we observed that curcumin led to a tendency to reduce soluble Aβ40 as well as to increase soluble Aβ42, and as a consequence the soluble Aβ42/40 ratio was significantly increased. Although a reduction in both soluble and insoluble Aβ has been previously described (Lim et al. 2001), those animals were chronically treated for 6 months with a different dosing and route. Our experiments were limited to 1 week of treatment, resulting in measurable clearance of deposited Aβ without large effects on biochemical measures of Aβ. It is intriguing that in our study we observed clearance of amyloid and reduced toxicity as measured by examining the structural alterations of neurites near plaques, despite the increase in soluble Aβ42/40 ratio which has been implicated as causative in AD (Walker et al. 2005). The animal model used in this work, APPswe/PS1dE9, has a higher Aβ42/40 ratio probably due to the PS1 mutation (Borchelt et al. 2002; Garcia-Alloza et al. 2006b), and the immediate effect of curcumin tends to exacerbate this tendency. Further studies with mouse models that favor the Aβ40/42 ratio could also help to elucidate this question. It is also possible that no changes in Aβ40 levels were detected because it might be more easily cleared.
We did not detect a statistically significant effect of curcumin on insoluble Aβ levels, despite the fact that a clear reduction in plaque size was observed after the treatment. A precedence exists for the lack of an effect on biochemical measures of Aβ following anti-Aβ therapy, despite improvements in behavioral tests (Janus et al. 2000). In our hands, this effect also suggests that longitudinal imaging provides a very powerful tool to detect amyloid clearance in vivo when other ex vivo or biochemical approaches are not sensitive enough to detect early changes. In total, it seems clear that there is an overall beneficial effect of curcumin in this and previous studies through modulation of Aβ (Shukla et al. 2003; Ono et al. 2004; Yang et al. 2005; Jung et al. 2006). Moreover, studies in rodents have shown that curcumin partially reversed behavioral abnormalities in APP mice (Frautschy et al. 2001) or after heavy metal induced neurotoxicity (Dairam et al. 2007).
Senile plaque deposition can be reduced by anti-Aβ antibodies (Bacskai et al. 2002b) and this effect seems to be at least partially mediated by microglial activation (Bard et al. 2000; Wilcock et al. 2003). However, previous studies with curcumin have shown its capacity to attenuate inflammatory response of brain microglial cells (Kim et al. 2003). In our hands microglia staining showed a classical distribution of microglial cells surrounding plaques without any apparent differences between curcumin treated and control mice. Although it is possible that the effects observed in cell culture cannot be directly extrapolated to in vivo studies, it can not be excluded that microglia are indirectly implicated in the clearance of plaques. On the other hand curcumin effects are highly dose-dependent in general and therefore it is possible that doses reaching the brain in the present study were not high enough for effects on microglia.
Abnormal neuritic curvature is observed in APPswe/PS1dE9 (Garcia-Alloza et al. 2006a) and in our hands curcumin showed a neuroprotective effect that led to a significant straightening of the distorted dendrites near and away from plaques. These effects suggest that aggregated Aβ as well as soluble Aβ species results in abnormal neuritic morphology that is reversible. However, we can not exclude an effect on inflammation or oxidative stress that may be responsible for the observed straightening effect. Previous studies have shown that abnormal neuronal morphology is correlated with the presence of Aβ and with neuronal dysfunction (Stern et al. 2004) and that anti-Aβ antibodies led to a rapid normalization of neuritic curvature (Lombardo et al. 2003). Other anti-oxidants also show a similar effect in vivo (Garcia-Alloza et al. 2006a), however, in contrast to anti-oxidant treatments, curcumin also reduces dystrophy size in close proximity to the plaques. This effect was demonstrated after anti-Aβ antibody treatment (Brendza et al. 2005), and may be an important determinant of an effective therapy. Although the anti-oxidant effects of curcumin cannot be excluded, other actions or a combination of actions of curcumin may be responsible for the effects observed on dystrophy size. It is also possible that the mechanisms underlying the alterations in trajectories and dystrophies may differ. Altogether, these data show that curcumin can prevent and reduce amyloid deposition in vivo and that it also partially restores dendritic abnormalities, suggesting that multifactorial curcumin effects can reverse a range of pathological features associated with AD. These studies provide support for the further elucidation of the mechanism of action of curcumin as a possible therapeutic treatment of AD.