Map kinase, extracellular regulated kinase (ERK) signaling and transcriptional activator cyclic-AMP response element binding protein (CREB) are required for memory formation in response to an influx of calcium [1
] as well as being involved in ischemia, oxyradical (ROS) stress, aging and neurodegeneration. For example, in neurodegenerative disease, the Alzheimer disease-associated peptide amyloid-beta (Aβ) stimulates MAP kinase ERK2 short-term while Aβ with ROS-promoting Fe+2
stimulates ERK2 long-term [2
]. Aβ alone [3
] or together with glutamate inhibits PKA and its downstream CREB target in embryonic neurons [4
]. In a human cell line, intracellular Aβ causes hyperphosphorylation of CREB to block nuclear translocation [5
]. This dichotomy between memory creation and disruption is not well understood. It is further complicated by age-related differences in memory, signal processing and susceptibility to ROS.
A cost-effective and palatable intervention against aging and neurodegeneration that promotes memory may be dietary blueberries, rich in phytochemicals. Under oxidative stress, polyphenols contained in tea, red wine, or ginkgo biloba affect cell signaling by altering extracellular signal regulated kinase (ERK) activity [6
], as well as reducing protein kinase C activity [8
] and decreasing CREB [10
]. Berries and fruit phytochemicals are well known for their antioxidant activities. Previously, we have shown that motor and cognitive deficits in aging could be reduced by feeding aged rats a diet containing 2% blueberries or strawberries [11
]. Subsequent research has supported these early findings, including a study showing that APP + PS-1 (amyloid precursor protein/presenilin-1) transgenic mice fed a diet containing 2% blueberry extract from 4–12 months of age showed no deficits in Y-maze performance when compared to mice fed an unsupplemented NIH-31 diet [12
]. Additionally, embryonic hippocampal neurons exposed to Aβ showed disruptions in calcium regulation that were prevented by pre-treatment of the cells with various fruit extracts [13
]. Because the reversals in whole animal studies could involve effects on the aging vasculature, inflammatory response, hormonal system, or neurons, whether similar protection is possible for isolated old neurons would further clarify the target.
Previous studies have shown that stressors such as Aβ can increase several additional transcription factors associated with oxyradical stress such as CREB [15
]. Moreover, acute hypoxia upregulates CREB [reviews 16
]. It has also been shown that CREB is activated by hydrogen peroxide in Jurkat T lymphocytes [18
] and by cadmium in mouse neuronal cells [19
] as well as during stroke [20
]. In a similar manner, PKCγ may be involved in the downstream activation of oxidative stress to activate CREB during protection by treatment with blueberry extract [13
]. From these studies, the relationship of Aβ and ROS to stress vs. memory signaling and neurotoxicity remains to be clarified.
We have developed a rat neuron model of aging in which neurons from old rats are cultured as easily as middle-age neurons in a common serum-free defined and optimized medium [21
]. As judged by immunostaining, these cultures of middle-age and old neurons are 80% neurons, 10% oligodendrocytes, 5% microglia and 5% astroglia, have the same amount of protein in their regenerated axons and dendrites, take up glucose at similar rates [22
] and have equal levels of resting respiration [23
]. Cultured middle-age and old neurons have similar passive membrane properties; both ages fire action potentials spontaneously [24
] and have similar resting membrane potential [25
]. Although the same numbers of neurons regenerate for these two ages, the old neurons are more susceptible to toxicity from glutamate, lactate or Aβ [21
]. The mechanism of cell death involves apoptosis subsequent to caspase activation and ROS generation [26
]. In this culture model of brain aging, we can determine whether the protection by blueberry extract in APP transgenic mice against Aβ toxicity and memory loss acts directly on the neurons specifically, avoiding the complexities of the vasculature, the inflammatory response, hormonal system or another uncontrolled target. Here, we determine whether blueberry extract is neuroprotective against Aβ toxicity in old neurons and which kinase pathway is associated the mechanism of toxicity and protection. We also determine whether blueberry extract lowers the resting rate of oxyradical production.