Gene and microRNA expression profiles in models of brain aging: effects of bioactive phenolic compounds

Summary of the results of in vivo experiments Age-related diseases have increasingly become the focus of much research interest in recent years, due to the progressive increase in human life expectancy in Western countries and, as a consequence, in the proportion of aged subjects in those populations. Aging is associated with illnesses of many human organs and tissues, but particularly important are those related to the aging of brain cells, which may underlie peculiar susceptibility to acute (e.g., stroke, traumatic events) and neurodegenerative diseases (e.g., Alzheimer’s and Parkinson disease) that represent a heavy economic burden and an unmet therapeutic need all over the world (Yanker et al., 2008) Understanding the mechanisms of age-related changes in the brain and identifying potential modulators may have profound consequences for the prevention and treatment of age-related impairments and diseases (Glorioso and Sibille, 2011). On the other hand, several studies have suggested a role for dietary and lifestyle factors in preventing or delaying, age-related diseases but more evidences to support a role of specific food components in brain aging, are still needed (Barberger-Gateau, 2014). The first aim of my research was to study the regulatory mechanisms of microRNAs on genes during the aging process in the brain, by integrating genome-wide miRNA and mRNA expression profiles. The second aim, was to verify whether dietary treatment with olive oil phenols modifies the profile of microRNA and gene expression in the aging mouse brain, and if these changes correlate with the cognitive and motor changes observed in the aging animals. C57Bl/6J mice fed from middle age to senescence with extra-virgin olive oil rich in polyphenols (H-EVOO, phenol dose/day: 6 mg/kg) showed cognitive and motor maintenance compared to aged-matched controls, fed with the same olive oil but deprived of the phenolic content (L-EVOO). We focused on two brain areas which are involved in cognitive and motor processes: cortex and cerebellum. Overall, our analyses demonstrated that after 6 months feeding, most of the gene expression changes, were restricted to the cerebral cortex, and for this reason, we later focused on this area alone. Dietary treatment with H-EVOO was associated with a significant modulation of genes (mostly up-regulated) compared to L-EVOO. Among those, we found Notch1, several bone morphogenetic proteins (BMPs), the nerve growth factor receptor (NGFR) the glucagon-like peptide-1 receptor (GLP1R) and CREB-regulated transcription coactivator 3 (CRTC3) previously associated to brain functioning, synaptic plasticity, motor and cognitive improvements. The agrin pathway, involved in cholinergic synaptic differentiation and maintenance, was also significantly modulated. We then analyzed global miRNA expression profiles in the cortex harvested from aged and young mice. Overall, H-EVOO-fed mice cortex displayed miRNA expression profiles similar to those observed in young mice. Sixty-three miRNAs, out of 1203 analyzed, were significantly down-regulated compared to the L-EVOO group; among them, mir-484, mir-27, mir-137, mir-30, mir-34 and mir-124. Evidence suggesting that these miRNA are potentially relevant to brain aging, comes from the finding that they are predicted to target the previously mentioned genes and actually, we found an inverse correlation with their target genes. Overall, these results indicate that olive oil phenols exert several beneficial effects in the brain by modulating signaling cascades which impact neuronal function and synaptic plasticity. The modulation of these genes and miRNAs can be thus considered among the molecular mechanism by which olive oil phenols exert their beneficial effects on memory and motor coordination in aging mice. Summary of the results of in vitro experiments In the last few years, the European Union strongly encouraged the “3Rs” principle (replacement, reduction and refinement) for the protection and spare of animals used for scientific purposes. In vitro systems, besides sparing animal lives, have the obvious advantage of being often less expensive and shorter in duration and offer a simplified model for shedding light into mechanisms involved in brain aging. Our purpose was to evaluate long-term neuro-glial co-cultures as a model for investigating senescence in the nervous system and to assess its similarities with in vivo models. To this aim, we maintained the cultures from 15 days in vitro (DIV) (mature cultures) up to 27 DIV (senescent cultures), measuring senescence-associated, neuronal, dendritic and astrocytic markers. Whole miRNA expression profiles were compared to those measured in the cortex of 18- and 24-month-old C57Bl/6J aged mice and of transgenic TgCRND8 mice, a model of amyloid-ß deposition. Neuro-glial co-cultures displayed features of cellular senescence (increased SA-β-gal activity, oxidative stress, γ-H2AX expression, IL-6 production, astrogliosis) that were concentration-dependently counteracted by the anti-aging compound resveratrol (1-5 µM). Besides recapitulating some characteristics of cellular senescence, neuro-glial co-cultures also display age-associated degenerative changes such as progressive neuronal loss and a decline in the dendritic network. Among the 1080 miRNAs analyzed, 335 were down-regulated or absent in 27 compared to 15 DIV and resveratrol reversed this effect. A substantial overlapping was found between age-associated changes in miRNA expression profiles in vitro and in TgCRND8 mice but not in physiologically aged mice, indicating that this culture model displays more similarities with pathological than physiological brain aging. These results demonstrate that neuro-glial co-cultures aged in vitro can be useful for investigating the cellular and molecular mechanisms of brain aging, and for preliminary testing of protective compounds.