Brain-targeted proanthocyanidin metabolites for Alzheimer's disease treatment

Abstract

While polyphenolic compounds have many health benefits, the potential development of polyphenols for the prevention/treatment of neurological disorders is largely hindered by their complexity as well as by limited knowledge regarding their bioavailability, metabolism, and bioactivity, especially in the brain. We recently demonstrated that dietary supplementation with a specific grape-derived polyphenolic preparation (GP) significantly improves cognitive function in a mouse model of Alzheimer's disease (AD). GP is comprised of the proanthocyanidin (PAC) catechin and epicatechin in monomeric (Mo), oligomeric, and polymeric forms. In this study, we report that following oral administration of the independent GP forms, only Mo is able to improve cognitive function and only Mo metabolites can selectively reach and accumulate in the brain at a concentration of ∼400 nM. Most importantly, we report for the first time that a biosynthetic epicatechin metabolite, 3'-O-methyl-epicatechin-5-O-β-glucuronide (3'-O-Me-EC-Gluc), one of the PAC metabolites identified in the brain following Mo treatment, promotes basal synaptic transmission and long-term potentiation at physiologically relevant concentrations in hippocampus slices through mechanisms associated with cAMP response element binding protein (CREB) signaling. Our studies suggest that select brain-targeted PAC metabolites benefit cognition by improving synaptic plasticity in the brain, and provide impetus to develop 3'-O-Me-EC-Gluc and other brain-targeted PAC metabolites to promote learning and memory in AD and other forms of dementia.

Figures

Figure 1.

Fractionation of GP and in vivo efficacy of Mo and Po on Aβ-related neuropathology in Tg2576 mice. A–C, Normal phase HPLC chromatograms of GP fractions: GP (A), Mo-enriched fraction (B), Po-enriched fraction (C). D–F, The influence of chronic Mo or Po treatment on Aβ-related spatial memory in Tg2576 mice using MWM test. D, Hidden platform acquisition; latency score represents the time taken to escape to the platform. E, Probe trial. Percentage of time in four different quadrants (T, target; O, opposite; R, right; L, left). F, Swimming speed. G–I, Quantifications of oligomeric Aβ (G), total Aβ1–42 (H), and Aβ1–40 (I) in brains of Mo-treated, Po-treated, or control (CTRL) mice using ELISA assay. Data represents mean ± SEM, n = 8–10 mice per group. *p < 0.05, **p < 0.01.

Figure 2.

Plasma pharmacokinetics, brain levels of C and EC metabolites, and structural characterization of biosynthetic EC metabolite. A, Plasma pharmacokinetic profile of major C and EC metabolites following repeated dosing of rats by treatment with GP, Mo, and Po. B, Concentration of C and EC metabolites in brain tissue following 10 d of treatment. Inset, LC-MS/MS separation of major C and EC metabolites detected in extracts of rat brain tissue collected after 10 d of treatment. MRM trace is shown for C/EC-O-β-glucuronide (465.1→289.1 m/z) and MeO-C/EC-O-β-glucuronide (479.1→303.1 m/z). Peak identifications: peak 1: (±)-C-O-β-glucuronide; peak 2: (-)-EC-O-β-glucuronide; peak 3: 3′-O-Me-(±)-C-O-β-glucuronide; peak 4: 3′-O-Me-(-)-EC-O-β-glucuronide. ***p < 0.001, n = 5 per group. C, Proposed structure of the primary EC metabolite identified as 3′-O-Me-(-)-EC-5-O-β-glucuronide present in blood and brain tissues following repeated dosing of rats by treatment with GP or Mo fraction. D, LC-MS-TOF separation and online spectra of C and EC metabolites detected in extracts of rat plasma (black) and biosynthetic EC metabolite (red). Extracted ion chromatogram is shown for MeO-C/EC-O-β-glucuronide (479.13). Peak identifications: peak 3: 3′-O-Me-(±)-C-5-O-β-glucuronide; peak 4: 3′-O-Me-(-)-EC-5-O-β-glucuronide; peak M: 3′-O-Me-(-)-EC-5-O-β-glucuronide.

Figure 3.

Biosynthetic 3′-O-Me-EC-5-O-β-glucuronide improves basal synaptic transmission and long-term potentiation coinciding with increased CREB hyperphosphorylation. A–D, The effect of 300 nm 3′-O-Me-EC-Gluc treatment on basal neuronal transmission and LTP in hippocampal slices from AD (A, B) and wild-type (Wt) mice (C, D). Arrow indicate the beginning of tetanus to induce LTP. E–I, The effect of 3′-O-Me-(-)-EC-5-O-β-glucuronide on CREB signaling pathway in hippocampal slices from old Tg2576 mice following 5 h 300 nm 3′-O-Me-EC-Gluc treatment. The levels of phosphor-proteins of CREB phosphorylation at Ser133 (E), Erk1/2 phosphorylation at Thr185/Tyr187 (F), MEK phosphorylation at Ser222 (G). H, I, PKA activity and protein content of PKA IIa subunit expression (H) and phosphor-CaMKII and total CaMKII (I) in brain slices from Tg2576 mice. Inset, representative Western blot image of P-CaMKII and total CaMKII. *p < 0.05, n = 5 per group.