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2008 SURI Intern Year: Junior Research Project: The Dose-dependent Effect of Phosphodiesterase 4D4 (PDE4D4) microRNA on Abeta1-42 induced Memory Deficits in Rats Introduction: Phosphodiesterase 4 (PDE4) is an enzyme that hydrolyzes cyclic AMP (cAMP) and plays a critical role in controlling its intracellular concentrations. PDE4 is an integral component of N-methyl-d-aspartate (NMDA) receptor-mediated cAMP signaling (Zhang et. al. 2000) suggesting that PDE4 in the hippocampus is important for cAMP signaling in the mediation of memory (Zhang et. al. 2004). By using lentiviral vectors harboring microRNAs (miRNA) specifically targeting PDE4D4, which is expressed in the hippocampus at relatively high levels, the expression of PDE4D4 will be down regulated, leading to an increase in cAMP concentrations. Cyclic AMP in turn phosphorylates CREB, as demonstrated by increased phosphorylated CREB (pCREB) levels. Activation of cAMP/CREB signaling increases memory-associated memory; it also increases hippocampal neurogenesis, which is contributed by PDE4 inhibition (Zhang et. al. 2008) and related to memory. Abeta1-42 is the most important beta amyloid peptide responsible for memory loss in Alzheimer’s disease. It has been shown that inhibition of PDE4 by rolipram reverses Abeta1-42 induced deficits of memory and cAMP signaling (Gong et. al. 2006). To determine whether PDE4D4 is involved in the effects of Abeta1-42, we will examine the dose-dependent effect of lenti-PDE4D4 miRNA on Abeta1-42 induced memory deficits in the step-through passive avoidance and object recognition tests in rats. The levels of pCREB also will be determined using immunoblot analysis. Methods: Male Sprague-Dawley rats (250-300 g) will be used in the experiments. 22-gauge guide cannulae will be implanted in bilateral CA-1 subregions of the hippocampus. The rats will then be injected via microinfusion with Abeta1-42 (4ug/side, iCA-1), followed by infusions of lenti-GFP, lenti-scrambled miRNAs (negative control), or lenti-PDE4D4 miRNAs at concentrations of 1, 4 , and 16 tu/side. After approximately 2 weeks, memory performance of rats will be tested using the step-through and object recognition tests. After completion of behavioral tests, animals will be sacrificed and the injection sites will be verified histologically. In addition, levels of pCREB and PDE4D4 in the hippocampus will be compared across groups using Western blots. The levels of PDE4D4 mRNA will also be tested using real time reverse transcription PCR (RT-PCR).
Gong B, Cao Z, Zheng P, Vitolo OV, Liu S, Staniszewski A, Moolman D, Zhang H, Shelanski M, Arancio O. Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory. Cell (2006) 126(4):775-788. Zhang HT, Crissman AM, Dorairaj NR, Chandler LJ, O’Donnell JM. Inhibition of cyclic AMP phosphodiesterase (PDE4) reverses memory deficits associated with NMDA receptor antagonism. Neuropsychopharmacology (2000), 23(2):198-204. Zhang HT, Huang Y, Masood A, Stolinski LR, Li Y, Zhang L, Dlaboga D, Jin SL, Conti M, O'Donnell JM. Anxiogenic-like behavioral phenotype of mice deficient in phosphodiesterase 4B (PDE4B). Neuropsychopharmacology (2008), 33(7):1611-1623. Zhang HT, Zhao Y, Huang Y, Dorairaj NR, Chanfler LJ, O’Donnell JM. Inhibition of the phosphodiesterase 4 (PDE4) enzyme reverses memory deficits produced by infusion of the MEK inhibitor U0126 into the CA1 subregion of the rat hippocampus. Neuropsychopharmacology (2004), 29(8):1432-1439.
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