Combination therapy in a transgenic model of Alzheimer's disease
Introduction
Alzheimer's disease (AD) is an incurable age-related neurodegenerative disease, characterized by progressive memory loss and irreversible cognitive decline. Aβ neurotoxicity is established to be a critical event in AD pathogenesis and correlated with dendritic spine loss, which causes synaptic failure resulting in cognitive dysfunction (Selkoe, 2008). Although multiple other complex systems are likely involved in the pathogenesis of AD, there is substantial evidence that Aβ neurotoxicity, especially non-fibrillar oligomers derived from Aβ42, is a fundamental event (Selkoe, 2004, Walsh and Selkoe, 2004). Many studies in both AD patients and animal models have shown that the brain levels of soluble oligomeric form of Aβ strongly correlate with senile plaques, one of the pathological hallmarks of AD (Koffie et al., 2009, Shankar et al., 2008). Therefore, many therapeutic strategies aiming to reduce Aβ accumulation and/or to accelerate its clearance are currently being developed in transgenic mouse models and cell culture models of AD.
In recent years, studies focused on cyclohexanols (inositols) have provided evidence that the scyllo-inositol interacts with Aβ42 peptide promoting its conformational change and stabilizing it in small, non-fibrillar complexes that are significantly less toxic to neuronal cells (McLaurin et al., 2000). In animal models of AD, scyllo-inositol was shown to reduce plaque burden, improve performance on memory tasks and restore hippocampal synaptic plasticity (McLaurin et al., 2006, Townsend et al., 2006). Scyllo-inositol is an orally bioavailable molecule that easily crosses the blood brain barrier and can be detected in the brain by magnetic resonance spectroscopy (MRS). Using this technique we could detect increased levels of scyllo-inositol in the hippocampus and frontal cortex in mice orally treated with scyllo-inositol in water (16.5 mg/L) (Choi et al., 2010a). Scyllo-inositol has recently been in human clinical trials for the treatment of patients with mild to moderate AD (Salloway et al., 2011). The effect of scyllo-inositol treatment on the concentration of Aβ42 in the cerebrospinal fluid (CSF) and on ventricular volume was evaluated in mild to moderate AD patients treated with 250 mg dose of scyllo-inositol twice daily for 78 weeks. Treatment with 250 mg dose of scyllo-inositol treatment significantly reduced CSF Aβ42 concentration compared to placebo group at 78 weeks. Moreover, scyllo-inositol at 250 mg dose showed a significant increase in ventricular volume measured by MRI (Salloway et al., 2011).
The protective effects of NSAIDs against AD pathologies are observed in various experimental models and NSAIDs have been one of the therapeutic approaches for AD treatment by either delaying onset or preventing progression of AD (Eriksen et al., 2003, in t' Veld et al., 2001, Jantzen et al., 2002, Weggen et al., 2001).
This approach has been supported in transgenic mouse models of AD with studies using subsets of NSAIDs such as ibuprofen and R-flurbiprofen to reduce amyloidogenic Aβ42 production (Eriksen et al., 2003, Kukar et al., 2007).
We have previously reported the beneficial effects of ibuprofen in lowering the Aβ levels in the triple transgenic mouse model of AD (3xTg-AD) (McKee et al., 2008) and in double transgenic Alzheimer mice (APPxPS1) (Choi et al., 2010b). Prophylactic treatment of 3xTg-AD mice with ibuprofen at 6 months of age showed a significant decrease in intraneuronal oligomeric Aβ and hyperphosphorylated tau immunoreactivity in the hippocampus, and prevented cognitive deficiency compared to untreated 3xTg-AD mice. In double transgenic mouse model of AD (APPxPS1) treatment with ibuprofen also provided significant protection against neuronal markers N-acetyl aspartate (NAA) and glutamate detected by MRS. In that study, we examined the effects of chronic NSAID, ibuprofen treatment on amyloid plaque deposition, Aβ peptide levels, and neurochemical profiles using MRS at two different ages in APPxPS1 mice. Ibuprofen significantly lowered the plaque burden in the 16- to 18-month-old mice as measured by the percent of cortical area. At 17 months of age, there was a decrease in the neuronal markers NAA and glutamate and an increase in the astrocytic markers glutamine and myo-inositol in APPxPS1 mice compared to age matched WT mice. Ibuprofen provided significant protection against NAA and glutamate loss, however it did not significantly affect myo-inositol or glutamine levels (Choi et al., 2010b).
Although the mechanism of the Aβ42 lowering NSAIDs has not been definitively established, the most convincing mechanism of the Aβ42 lowering activity of selected NSAIDs suggests a direct allosteric modulation of γ-secretase activity. This action of these NSAIDs was independent of their COX activity (Weggen et al., 2001).
R-Flurbiprofen is an enantiomer of flurbiprofen, a class of NSAIDs. R-Flurbiprofen practically lacks COX-activity and in humans undergoes very limited chiral inversion to the S-enantiomer that maintains its activity against COX (Geisslinger et al., 1994, Wechter et al., 1994). This enables the use of high levels of R-flurbiprofen in the absence of COX mediated side effects. It has been reported that chronic administration of R-flurbiprofen attenuates the spatial learning deficits if given prior to plaque deposition in Tg2576 mice (Kukar et al., 2007).
However, the clinical results from phase III trial of tarenflurbil (formerly R-flurbiprofen) failed to slow cognitive and functional decline over 18 months in patients with mild Alzheimer's disease (Green et al., 2009).
AD remains an incurable disease. Despite promising results in preclinical studies over the past several years, clinical trials have failed to show efficacy in AD patients. The combination of pharmacological therapies has been successfully developed for the treatment of several diseases, including cancer and diabetes. We designed this study to test the hypothesis that the combination therapy of scyllo-inositol with R-flurbiprofen would have an additive effect and further ameliorate and/or reverse amyloid pathology and cognitive decline in 5XFAD mice.
Section snippets
Mice
5XFAD transgenic mice, developed by Oakley (Oakley et al., 2006), coexpress human APP and PS1 by combining multiple FAD mutations [APP K670N/M671L (Swedish) + I716V (Florida) + V717I (London) and PS1 M146L + L286V] with neuronal expression driven by the Thy-1 promoter were purchased from Jackson Laboratory. WT mice from 129/C57BL6 background strain were used as control animals. Only female mice were used in the current study (10 mice per group). All animal experiments were carried in accordance with
Effect of scyllo-inositol alone and in combination with R-flurbiprofen treatments on spatial learning memory
Treatment of 5XFAD mice with scyllo-inositol or scyllo-inositol + R-flurbiprofen combination started at 7 months of age and continued for one month. No side effects were observed to either treatment. Body weights of 5XFAD treated mice, untreated 5XFAD and WT mice were comparable at all times. At 8 months of age, spatial learning and memory were tested in a RAWM. In this test, mice are trained in 4 consecutive trials per day (T1-T4) for 60 s each to escape onto a submerged platform placed at the end
Discussion
Imaging studies have shown that Aβ plaques start to accumulate prior to onset of clinical symptoms and drugs currently being developed mostly target Aβ for prevention of AD. In view of the potential benefit of scyllo-inositol inhibiting the aggregation of Aβ into oligomers and Aβ neurotoxicity, we proposed combining scyllo-inositol with a complementary therapy, which targets Aβ through a distinct mechanism to further increase the beneficial effects of scyllo-inositol. To test this concept, we
Conclusions
These results show that scyllo-inositol treatment lead to significant reduction in the Aβ plaque burden in the brains of 5XFAD mice, protected the decrease in NAA, GABA and glutamate and improved memory thus providing a rationale for the potential benefit of the use of scyllo-inositol in treatment of AD. However, treatment with scyllo-inositol combined with R-flurbiprofen was overall not more effective to decrease Aβ levels and plaque burden or protect against neurochemical changes when
Acknowledgments
This research is supported by grants from NIA (R01AG031896) to A. Dedeoglu and the Department of Veteran Affairs (Merit Award) to A. Dedeoglu. The authors thank Lokman Hossain for animal husbandry, and Dr. Kenton H. Zavitz for providing the R-flurbiprofen (tarenflurbil).
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Contributed equally.