Nutraceutical approaches against amyloid-β induced neuropathology: an in vivo and in vitro study.

ABSTRACT Introduction: Mounting evidence supports the beneficial effects of the Mediterranean diet (MD) and the Asian diet in delaying ageing and in preventing age-related dysfunctions, cancer, diabetes and neurodegenerative diseases. The beneficial effects of the MD and Asian diets in reducing age-related dysfunctions, including Alzheimer’s disease (AD), could be the consequence of the presence in specific foods of substantial amounts of specific polyphenols whose beneficial properties include the ability to interfere with amyloid aggregation. Our previous data have highlighted the beneficial effects of oleuropein aglycone (OLE) against protein/peptide aggregation in vitro and in TgCRND8 (Tg) mice, a model of Aβ deposition. Aim: To check in Tg mice 1) the effects of OLE or a mix of polyphenol extracts or hydrohytyrosol (HT) on cognitive functions and neuropathology and 2) the effect of andrographolide administration on cognitive functions and its ability to cross the blood brain barrier (BBB) conjugating it with fluorescent albumin nanoparticles. In addition, in rats we investigated 1) the ability of intravenous (i.v.) administered human fetal cholinergic neurons (hfCNs), isolated from the nucleus basalis of Meynert (NBM) of 12-week old fetuses, to cross the BBB and to improve memory functions of quisqualic acid NBM-lesioned rats and 2) the ability of intravenously administered solid lipid nanoparticles (SLNs) to cross the BBB and to penetrate the central nervous system of adult rats. Methods: In vivo experiments were carried out using: 1) Tg and wild type (wt) mice treated for 8 weeks with a modified low-fat (5.0%) AIN-76A diet (10 g/day/ mouse) as such, supplemented with OLE (50mg/kg of diet) or with a mix of polyphenol extracts (50 mg/kg of diet) found in olive mill waste water or with HT (50 mg/kg of diet), 2) Tg and wt mice injected intraperitoneally (i.p.) for 4 weeks (3 i.p. for week) with andrographolide (ANDRO) (4 mg/kg) conjugated with fluorescent albumin nanoparticles (NPs-ANDRO), 3) male Wistar rats divided into the following groups: Group I: injected into the NBM with 0.5 μL of quisqualic acid and subsequent intravenous (i.v.) administration by the tail vein of 300 μL of 1.5ML hfCNs. Group II: injected into the NBM with 0.5 μL of quisqualic acid. Group III: injected into the NBM with 0.5 μL saline and subsequent i.v. administration by the tail vein of 300 μL of 1.5M hfCNs. Group IV: injected into the NBM with 0.5 μL saline; Group V: un-injected rats and 4) male Wistar rats divided into the following groups Group I: injected i.v. with 200 L of nanoparticles (SLN 28.57 mg/ml plus FITC 1.43 g/ml) and sacrificed after 3h; Group II: injected i.v. with 200 L of nanoparticles and sacrificed after 24h; Group III: injected i.v. with 200 L of nanoparticles and sacrificed after 72h; Group IV: injected i.v. with 200 L of 0.9% of saline. For in vitro experiments were used neuroblastoma cell line (N2a) maintained in a 5.0% CO2 humidified atmosphere at 37°C. Results and Conclusion: Our results show that OLE supplementation induces epigenetic modifications as previously reported, favoring the expression of SIRT1 and reducing that of PARP1 confirming the existence of a functional link between PARP1 and SIRT1. Furthermore, diet supplementation with a mix of polyphenols or HT strongly improved mouse cognitive performances and reduced ß-amyloid deposits. An intense activation of autophagy was found in the cortex of all treated groups. HT administration also affected the inflammatory response in the hippocampal areas, as shown by the reduced astrocytes activation and TNF-α mRNA levels. These results show that diet supplementation with a mix of polyphenols or HT at the same dose as that of pure OLE previously administered ameliorate cognitive functions and indicate that either a mix of polyphenols or HT treatment is useful to treat neurodegenerative diseases. In addition, we found that i.v. administration of hfCNs migrated to the injected NBM and along the needle tract. In the quisqualic acid NBM-injected rats the cognitive impairments were significantly improved by hfCNs. Finally, we found that both SLNs and NPs were able to cross the BBB and that NPs-ANDRO were able to cross the BBB and to improve cognitive functions in Tg mice, supporting that the developed nanocarriers represent new potential brain delivery system to increase the efficacy of andrographolide treatment in neurodegenerative diseases.