Location: Boston, Massachusetts2010 Annual Report
1a. Objectives (from AD-416)
1: Determine the effects of SB supplementation on behavioral aging using paradigms sensitive to cognitive (short and long-term memories) behaviors. 2: Determine the effects of SB supplementation on calcium dependent and neuronal signaling (2) and neurogenesis (2b) correlate these with alterations in behavioral parameters determined in SA 1. 3: Determine whether the efficacies of these supplementations in the behavioral assays are associated with enhanced resistance to oxidative stress or inflammation. 4: Establish the effects of nutritional modulation (e.g., berry fruit) on behavioral (e.g., cognition, gait, force and balance) and neuronal deficits in aging to assess the mechanisms involved and the most effective dietary supplements in animal and human models.
1b. Approach (from AD-416)
Aim 1. The effects of SB dietary supplementation (2% SB extract in the diet for 8 weeks. After 8 weeks performance will be examined in the rats using age-sensitive cognitive behaviors that are selective for reference and working memories. The latter behaviors will be assessed using the Morris water maze and the radial arm water maze, as well as novelty tests. All of these tests have been validated as being age sensitive (e.g. demonstrated a significant decline as a function of age). Aim 2. The effects of SB dietary supplementation on neurogenesis and differentiation will be examined using immunocytochemistry, bromodeoxyuridine (BrDU) incorporation in hippocampus and olfactory bulb obtained from the supplemented behaviorally-assessed animals Calcium signaling will be assessed by examining Ca45 clearance in tissues (as above) taken from the brain. Aim 3. For the basal assessments we will use immunohistochemistry to assess various markers of oxidative stress (e.g., HO-1) and inflammation (e.g., cytokines) as well as immunoblotting to detect HO-1 and bcl 2 expression. The responses of the tissue (muscarinic receptor sensitivity, HSP-70 activation) to oxidant (hydrogen peroxide, 10 uM) or inflammatory (LPS) stressors will be assessed by exposing cross-cut slices of the various brain regions obtained from the supplemented animals. Conduct studies using a specialized Noraxon treadmill in order to measure parameters of gait including balance, stepping up and down, and walking patterns in humans. The recently developed hardware by Noraxon will allow us to focus on more subtle variations in gait that include stride to stride fluctuations (e.g., symmetry) and associated alterations in electromyography (EMG). The Noraxon measurement system allows an objective kinematic analysis of the human gait by means of analyzing the tracks of body surface markers. Additionally, cognition will be measured with a battery of tests that measure memory and vigilance.
3. Progress Report
Previously, we have shown that whole, crude berry extracts are able to reverse several parameters of brain aging, as well as age-related motor and cognitive deficits when fed to rats from 19-21 months of age. These effects may be the result of direct effects on brain signaling or indirect effects through antioxidant and anti-inflammatory properties of the polyphenols. This year we attempted to extend these findings by attempting to determine the mechanisms of the beneficial effects of the strawberries. In this respect, we showed that strawberry 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 strawberry 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. 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.