Consumption of pomegranates improves synaptic function in a transgenic mice model of Alzheimer's disease

Abstract

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder characterized by extracellular plaques containing abnormal Amyloid Beta (Aβ) aggregates, intracellular neurofibrillary tangles containing hyperphosphorylated tau protein, microglia-dominated neuroinflammation, and impairments in synaptic plasticity underlying cognitive deficits. Therapeutic strategies for the treatment of AD are currently limited. In this study, we investigated the effects of dietary supplementation of 4% pomegranate extract to a standard chow diet on neuroinflammation, and synaptic plasticity in APPsw/Tg2576 mice brain. Treatment with a custom mixed diet (pellets) containing 4% pomegranate for 15 months ameliorated the loss of synaptic structure proteins, namely PSD-95, Munc18-1, and SNAP25, synaptophysin, phosphorylation of Calcium/Calmodulin Dependent Protein Kinase IIα (p-CaMKIIα/ CaMKIIα), and phosphorylation of Cyclic AMP-Response Element Binding Protein (pCREB/CREB), inhibited neuroinflammatory activity, and enhanced autophagy, and activation of the phophoinositide-3-kinase-Akt-mammalian target of rapamycin signaling pathway. These neuroprotective effects were associated with reduced β-site cleavage of Amyloid Precursor Protein in APPsw/Tg2576 mice. Therefore, long-term supplementation with pomegranates can attenuate AD pathology by reducing inflammation, and altering APP-dependent processes.

Keywords: Gerotarget; amyloid beta protein; amyloid precursor protein; inflammation; pomegranates; synapse.

Figure 1. Synaptic structural proteins in brain homogenates detected by Western blot analysis

The levels of PSD-95, Munc18-1, SNAP25, synaptophysin, p-CaMKIIα/ CaMKIIα, and pCREB/CREB in the brains of mice fed 4% pomegranate diet for 15 months. Treatment I: Wild type (non-transgenic) control of the APPsw mice fed with regular diet; Treatment II: APPsw mice also fed with regular diet; and Treatment III: APPsw mice fed with 4% pomegranate fruit diet. A. The blot shown is representative tracings of an experiment done six times. B. Graphs are mean ± S.E brains from tissue obtained from six rodents for each treatment group. Each bar of the quantification graph represents the corresponding band for each age group. Significance ^*p < 0.01 compared to wild-type mice fed with regular diet, ^#p < 0.01 compared to APPsw transgenic mice fed with regular diet.

Figure 2. mRNA expression of genes encoding for neurotrophic factors and proinflammatory markers

Treatment I: Wild type (non-transgenic) control of the APPsw mice fed with regular diet; Treatment II: APPsw mice also fed with regular diet; and Treatment III: APPsw mice fed with 4% pomegranate fruit diet. The levels of two important neurotrophic factors, BDNF and IGF-1, and the proinflammatory cytokines, tnf-α, il-1β, iNOS, ccl2, and il-10, in the brains of mice fed 4% pomegranate diet for 15 months were determined using real-time polymerase chain reactions. Graphs are mean ± S.E brains from tissue obtained from six rodents for each treatment group. Significance ^*p < 0.01 compared to wild-type mice fed with regular diet, ^#p < 0.01 compared to APPsw transgenic mice fed with regular diet.

Figure 3. Protein expression of autophagic markers following long-term supplementation with 4% pomegranates in APPsw/Tg 2576

The levels of autophagic markers, LC3 and bcl1, in the brains of mice fed 4% pomegranate diet for 15 months were determined using western blot analysis. Treatment I: Wild type (non-transgenic) control of the APPsw mice fed with regular diet; Treatment II: APPsw mice also fed with regular diet; and Treatment III: APPsw mice fed with 4% pomegranate fruit diet. A. The blot shown is representative tracings of an experiment done six times. B. Graphs are mean ± S.E brains from tissue obtained from six rodents for each treatment group. Each bar of the quantification graph represents the corresponding band for each age group. Significance ^*p < 0.01 compared to wild-type mice fed with regular diet, ^#p < 0.01 compared to APPsw transgenic mice fed with regular diet.

Figure 4. mTOR signaling pathway in brain homogenates detected by Western blot analysis

The levels Akt, mTOR and p70S6K, and their phosphorylated forms in the brains of mice fed 4% pomegranate diet for 15 months. Treatment I: Wild type (non-transgenic) control of the APPsw mice fed with regular diet; Treatment II: APPsw mice also fed with regular diet; and Treatment III: APPsw mice fed with 4% pomegranate fruit diet. A. The blot shown is representative tracings of an experiment done six times. B. Graphs are mean ± S.E brains from tissue obtained from six rodents for each treatment group. Each bar of the quantification graph represents the corresponding band for each age group. Significance ^*p < 0.01 compared to wild-type mice fed with regular diet, ^#p < 0.01 compared to APPsw transgenic mice fed with regular diet.

Figure 5. BACE1 and APP processing in brain homogenates detected by Western blot analysis

The levels APP, BACE1, CTFα, CTFβ, sAPPβ, ADAM10 and ADAM17 in the brains of mice fed 4% pomegranate diet for 15 months. Treatment I: Wild type (non-transgenic) control of the APPsw mice fed with regular diet; Treatment II: APPsw mice also fed with regular diet; and Treatment III: APPsw mice fed with 4% pomegranate fruit diet. A. The blot shown is representative tracings of an experiment done six times. B.Graphs are mean ± S.E brains from tissue obtained from six rodents for each treatment group. Each bar of the quantification graph represents the corresponding band for each age group. Significance ^*p < 0.01 compared to wild-type mice fed with regular diet, ^#p < 0.01 compared to APPsw transgenic mice fed with regular diet.