2010 Annual Report 1a.Objectives (from AD-416) To determine the dose and duration effects of the wild blueberry powder (WBP) on age-related differences in resistance to oxidative stress/inflammation (OS/INF) by exposing young and old animals to lipopolysaccharide (LPS). Brain regional differences in stress signaling (e.g,, nuclear factor kappa B, Nf'B), cytokine activation (e.g., TNFa), and OX-6, a marker of microglial activation, as well as differences in microglial morphology, will be assessed. Tissue levels of the polyphenols will be assessed following the various doses and durations of WBP to examine bioavailability. 1b.Approach (from AD-416) AIM 1 - The effects of the age-related differences in resistance to inflammation will be assessed by supplementing (for 2, 4, or 8, weeks) young (4 mo) and old (19 mo) animals with a control diet or a diet containing the equivalent of 0, ½, or 1.5, 4.5 cups/day (in human terms) of WBP. One half of each group of animals will then be administered a vehicle (saline) or LPS (5 mg/kg i.p.) just before they euthanized and stress (e.g., Nf'B, cytokines) and protective (ERK) signals assessed (Figure 1). We will also determine levels of OX-6, a marker of microglial activation, as well as differences in microglial morphology. A subset of animals will be euthanized at each time-point for assessment of stress signaling and tissue levels of the polyphenols. AIM 2 - We will also compare and correlate the degree of alterations in the various signals to the rats’ behavioral (e.g., radial arm water maze, and various motor tests e.g., rotorod) performance. Additionally, using bromodeoxyuridine (BrdU) to identify areas of the dentate gyrus showing increased DNA incorporation (an indicator of cell proliferation) and neuronal (Ngn2) and glial (GFAP) markers, we will determine differences in neurogenesis among the various groups. Dr. Kalt will measure tissue levels of polyphenols to assess bioavailability of the various WBP doses. 3.Progress Report Previously, we found that one of the major effects of wild and cultivated blueberry (BB) application to cells may be to alter oxidative stress/inflammatory signaling. This year we attempted to extend these findings by attempting to determine additional mechanisms of the beneficial effects of the blueberries. In this respect, we showed that blueberry extracts were able to rescue neurons through induction of autophagy, a process by which toxic debris is recycled and cleared in neurons. This is important since if the neuronal cell’s ability to clean up and remove toxic debris is antagonized by oxidative or inflammatory stressors, the cell may lose viability and show declines in function. The finding that blueberry pre-treatments can mitigate these effects has important implications for preventing declines in neuronal function via nutrition. It appears that the mechanisms involved in these beneficial effects may also involve declines in the signals that are enhanced by the stressors. Thus, we also began to determine the effects of the berry fruit on stress signaling from different types of brain cells such as microglia, astrocytes, and neurons derived from the mixed cultures. We showed that blueberries (BB) and strawberries (SB) could protect neurons through auxiliary effects on astrocytes when they were co-cultured with the neurons, as they are in the brain. Specifically, pretreatment with BB, SB, or SB+BB combinations reduced the inflammatory and stress protein levels in astrocytes, thereby exerting the protective effect. These berry extracts also increased protective proteins in both neurons and astrocytes, thus showing that mechanisms other than the inactivation of free radicals (molecules producing oxidative stress) are involved in the beneficial effects of the berry fruits. Additionally, we began to examine individual components from blueberries to determine which polyphenolic families might be responsible for their beneficial effects. In an animal study, we compared the effects of specific compounds found in blueberries with the effect of the whole berry on the mobility and behavior of aged rats in doses equivalent to 2 cups/day in humans. The findings suggested that proanthocyanidins may be the polyphenolic family responsible for the protective effects of blueberries; however, the combination of polyphenols present in blueberries may have a synergistic effect which provides increased protection from age-related declines in mobility and cognition relative to individual constituent compounds. These findings are important in both industry and health areas, since they point once again to the importance of including berries in the diet for promoting healthy aging. Monitoring of this project is accomplished through e-mail and/or teleconference.