Submitted to: Journal of Alzheimer's Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/17/2006
Publication Date: 3/1/2006
Citation: Joseph, J.A., Fisher, D.R., Bielinski, D. 2006. Blueberry extract alters oxidative stress-mediated signaling in cos-7 cells transfected with selectively vulnerable muscarinic receptor subtypes. Journal of Alzheimer's Disease. 9(1):35-42. Interpretive Summary: A great deal of research has indicated that one of the hallmarks of aging and neurodegenerative diseases of aging such as Alzheimer Disease (AD) is an increased sensitivity to very reactive molecules called free radicals. Free radicals, because many of them utilize oxygen, produce oxidative stress (OS) which can have a variety of negative effects on the brain and elsewhere in the body. In addition, studies have shown that a peptide called amyloid beta, which forms the hallmark plaques in the AD brain may alter the regulation of calcium in cells such that the cells cannot expel the calcium that enters the cells upon stimulation. This produces even more OS and could lead eventually to a loss of function in neuronal cells, as has been observed in aging and diseases such as AD. Increasingly, it also appears that dietary factors such as fruit and vegetable intake may play a role in preventing the onset of this disease. Based upon our previous work in transgenic mice that show AD-like changes in the brain and behavior, we found that blueberries might be effective in this regard. Recently, we showed in cells that different subtypes of neurotransmitter receptors called Muscarinic receptors, which are important in the regulation of calcium, respond to agents which produce OS. Some of the Muscarinic receptor subtypes when placed in cells increased the sensitivity agents which produce OS, such as dopamine or amyloid beta. This caused the cells to lose their ability to regulate calcium levels when they were stimulated. In this study we used molecular biological techniques to assess the various signals that are generated during Muscarinic receptor stimulation following OS. We also examined the effectiveness of blueberry extract in preventing these OS effects. The results suggested that the differences in OS sensitivity may involve differential brain cell signaling in a group of molecules called mitogen activated protein kinases (MAPK), and cyclic adenosine monophosphate response element binding protein (CREB) under OS-treatment conditions, suggesting that the native protection in these receptors against OS and inflammation may be derived from reduced activation in CREB. These findings also suggest that blueberries may antagonize OS effects by lowering activation of CREB, and possibly other OS-initiated signals. Taken together with our previous findings in animals it appears that Berryfruits such as blueberries may have dual effects on the MAPK pathway that depend upon the stimulus i.e., increasing the activity of MAPK and CREB during learning and memory and decreasing them under conditions of OS and possibly, inflammation.
Technical Abstract: Previous research has indicated that selective vulnerability (SV) to oxidative stress (OS) may be important in determining regional differences in functional declines in neuronal aging. OS SV may involve selective deficits in Ca2+ buffering (Ca2+ recovery time following oxotremorine application) to OS, determined in-part by Muscarinic receptor (MAChR) subtype with M1, M2 and M4AChR showing greater OS-induced loss [via dopamine (DA) exposure for 4 hrs] of Ca2+ recovery time than that seen in M3 or M5 cells. Deficits were antagonized by pre-treating M1, M2, or M4 AChR-transfected cells with blueberry (BB) extract. Thus, we assessed whether these OS SV differences might involve differential patterns of DA-induced protein kinase isoforms (PKC', PKC') and/or cyclic AMP response element binding protein (CREB) activation, and whether these differences might be altered by BB treatment. M1 or M3 AChR-transfected COS-7 cells were exposed to 1mM DA, and activation of phospho- (p) mitogen-activated protein kinase (MAPK) signaling was examined by immunoblotting and immunofluorescence analyses. The results showed that DA increased phosphorylated pCREB and pPKC' for both M1- and M3-transfected cells, and BBs decreased these DA-induced alterations, when measured by immunoblotting techniques. This same pattern was also observed for pPKC' among the various conditions in both transfected cell types, and for pCREB for M1, when fluorescent imaging was utilized to analyze these kinases. DA up-regulated CREB significantly less in M3- than in M1-transfected cells. Taken together these findings suggest that M1/M3 oxidative stress sensitivity differences may involve differential signaling in pMAPK and pCREB under OS-treatment conditions, suggesting that the native protection in these receptors against OS and inflammation may be derived from reduced activation. These findings also suggest that BB may antagonize OS effects induced by DA in M1-transfected cells by lowering activation of pCREB, and possibly pPKC'.