Early findings suggested that there was significant neuronal loss and other morphological changes that occur in the aging brain (22
). However, additional studies indicated that the age-related changes are much more subtle and involve calcium dysregulation (23
). While these age-associated changes appear to be dependent upon the particular neurons involved, they all involve various changes in calcium homeostasis and alterations in calcium regulation, with hippocampal and cortical neurons showing the greatest alterations (26
). As indicated in the Introduction, however, all of the changes involve some decline in calcium buffering. It has been postulated that these changes are the result of oxidative stress (28
Indeed, results of the present paper would support these findings, since DA, Aβ42, and LPS all reduced calcium buffering in the hippocampal cells. However, it appeared that, overall, the protective effects of the various fractions on calcium buffering were dependent upon the particular stressor to which they were exposed. Generally, the whole BB extract and pre-C18 fraction were the most effective in protecting CAR among the stressors, especially with regard to DA and Aβ42, while CA offered the least protection. These findings suggest that it may be the synergistic effects among the various polyphenolic families in the least fractionated forms of the blueberry (e.g., BB and PRE-C18) that provided the most generalized protection against DA and Aβ42. However, note that there were differences between the BB extract and the PRE-C18 fraction in protecting CAR against LPS. It appeared that CAR was somewhat impaired when LPS was applied to the PRE-C18 treated cells. This was not seen with the BB extract-treated cells, where CAR decreases were not seen with LPS. These differences were also reflected in the assessments of Viability where it appeared that the PRE-C18 fraction lowered viability in the absence of the stressors and a further lowering was seen with LPS treatment. Again these alterations in viability were not seen in the cells treated with the BB extract, although there were no differences in viability in DA- or Aβ42-treated cells that were pre-treated with the BB extract or the PRE-C18 fraction. There was also some indication that the PRE-C18 fraction was not as protective against increases in ROS with DA- or Aβ42. These differences could be the result of differences in concentrations between the two pre-treatments (BB or PRE-C18), since the concentrations utilized were based upon the phenolic levels of the compounds in the whole BB. The water soluble BB extract was utilized at 500µg/ml while the PRE-C18 fraction utilized was one-half that of the BB extract (250 µg/ml). These differences could also reflect source variations, since the BB extract was derived from frozen whole Tifblue cultivated BBs, while the PRE-C18 fraction was derived from wild blueberry juice. The types of phenolics in the two fractions may differ, making direct comparisons between BB and PRE-C18 difficult. However, since we have previous data using the Tifblue BBs it was necessary to add this condition as a positive control. Additionally, the Vaccinium angustifolium berries used to produce the juice included literally many hundreds, and possibly thousands, of genotypes harvested from the semi-cultivated wild stands. This complex mixture would therefore represent the average phenolic composition for the species. This is in contrast to cultivated blueberries (Vaccinium virgatum was used in this study), whose commercial juice may be produced from far fewer genotypes and, for that reason, may be subject to genotypic variability within the species.
Among the fractions, PAC, the high molecular weight (HMW) proanthocyanidins, and POST-C18 fractions were not effective against DA in protecting CAR. However, except for CA which was not effective with any of the stressors, all of the fractions were significantly more protective when LPS or Aβ42 was used as the stressor. In fact, overall, fewer differences in protection among the fractions were observed with LPS or Aβ42 than that seen with DA.
DA oxidation may be involved in the neuronal toxicity in neurodegenerative diseases such as Parkinson disease, where DA is easily oxidized to form DA quinones and other ROS species such as 3,4-dihydroxyphenylacetaldehyde or 3,4dihydroxyphenylethyleneglycolaldehyde (DOPEGAL), and may be responsible for neuronal loss in Parkinson disease (29
). One could speculate that reductions of ROS via nutritional supplementation with BBs may reduce DA toxicity. In this respect, in the present experiments when ROS was assessed among the various fractions, the data showed that even in the absence of the stressors (DA, LPS or Aβ42
) some of the fractions raised rather than lowered ROS. These included the HMW, LMW, ANTH and CA. These findings support previous research which has shown that plant polyphenols can act as potent pro-oxidants. For example, research has indicated that resveratrol (30
), flavonoids in general (31
), tannins (32
) and curcumin (32
) can increase ROS.
While it has been suggested that the pro-oxidant effect of plant polyphenols may be an artifact (33
) of in vitro methods, it is clear from the present paper that not all of the fractions, at least at the levels assessed here, increase ROS. It is possible that higher concentrations of these fractions may induce ROS increases. These include PAC, POST-C18 and to some extent the PRE-C18 fractions, in addition to the whole BB. As alluded to above these findings suggest that even in vitro the less fractionated that the fruit extract is, the less likely that ROS generation is seen in the absence of the stressors.
What is even more important is that when one considers the use of polyphenolic fractions as antioxidants in the presence of the various stressors, the pro-oxidant effects of the treatments may be even greater than that seen with the stressors alone. In the case of Aβ42
only the whole BB and the PAC treatments reduced the Aβ42
–induced ROS effects; virtually all of the other fractions acted synergistically with the Aβ42
, while HMW, LMW and ANTH fractions all acted synergistically with DA to increase ROS. Similar pro-oxidant synergistic effects have been reported previously with plant polyphenols (34
However, when the parameters of calcium buffering and ROS are considered together, except possibly for CA treatment in the presence of LPS or Aβ42
, there appears to be little relationship between ROS generation in the presence or absence of the stressor and the ability of the various fractions to mitigate the effects of the stressors on calcium buffering. As mentioned above, most of the fractions were protective, at least under the LPS or Aβ42
treatment conditions. These dichotomies suggest that ROS effects may not be reflective of the beneficial effects of the fractions on calcium buffering. It may be that their protective effects on calcium buffering involve alterations in downstream stress signals rather than direct quenching effects on ROS. We have shown in a previous study that the BB extract decreases several stress signals such as calcium response element binding protein (CREB), protein kinase Cγ (PKCγ), and P38 MAPK, among other stress mediators that were enhanced by DA application to hippocampal cells (16
). In the present experiment, DA, BB, ANTH and PRE-C18 significantly raised pMAPK over that seen in control cells, and each of the fractions (except for ANTH) increased pMAPK in the presence of DA.
BB reductions in pJNK in the presence of DA were greater than those of the other fractions, while PAC and POST-C18 increased pJNK in the presence of DA. Although BB and all the fractions increased pNFκB, in the absence of DA only the whole BB reduced this parameter to a value lower than the control value. Similar findings were seen with DA and BB treatment with respect to pP38 MAPK where DA alone slightly increased this stress signal but BB prevented the DA-induced increases in pP38 MAPK to a greater extent than any of the fractions. Thus, some of the differences between BB extract and the PRE-C18 fraction that were discussed above are also reflected in stress signal assessments, where DA-induced increases in JNK was greater with PRE-C18 pre-treatment than with the BB extract. However, the findings with respect to the other stress signals and pMAPK were similar.
However, despite these differences, taken together these data suggest that that the major protective effects of the whole BB extract and to some extent the PRE-C18 fraction involve the reductions of DA-induced increases in stress signal activation (pP38 MAPK, and pNFκB). Additional evidence for this hypothesis regarding the various berry extract and fraction pre-treatments can be seen when the viability and ROS parameters are compared. In some cases there were parallels between the two parameters (e.g., ANTH increases in ROS under stressor conditions with corresponding decreases in viability, LMW under DA and LPS conditions with decreases in viability, and PAC with decreases in viability and increases in ROS with Aβ42 and LPS) but in other cases there was a dichotomy between the two dependent measures (e.g., HMW increases in ROS with Aβ42 and DA with no decreases in viability; increases in ROS with PRE-C18 and POST-C18 treatments with no decreases in viability with Aβ42), suggesting alternative forms of protection that may have protected CAR and also prevented losses of viability.
When viability was assessed in the hippocampal cells in the absence of the stressors, some of the fractions decreased viability (LMW, PRE- and POST-C18). One of the most interesting findings concerning viability is that, as with ROS, there seemed to be a dichotomy between the effects of some of the fractions on viability (e.g., CA) and the level of protection provided by these fractions on hippocampal cell calcium buffering (CAR). For example, in the presence of DA there were differences among the fractions regarding the level of protection of CAR. However, there were fewer differences in the efficacy of the fractions when Aβ42
or LPS were used as stressors. This was not seen with viability where there were differences among the fractions regardless of the particular stressor utilized. This would suggest that assessments of viability may not be related to changes in CAR, and the cell, while showing decreases in viability, may still function with respect to CAR as a normal cell. This dichotomous condition is similar to that seen in normal aging where viability does not seem to explain losses in CAR. For example, it has been suggested that the degree of disruption of calcium buffering may not alter cell function or even viability, but it does make the cell more vulnerable to other stressors (37
). As mentioned above, in the present study, chlorogenic acid was not protective against CAR decreases induced by any stressor, but offered some protection against viability loss with these stressors. Clearly, however, these relationships are complex and are stressor/fraction/parameter dependent.
Additionally, previous mechanistic studies have also shown that, in addition to their potent antioxidant/anti-inflammatory effects, the beneficial properties of fruit polyphenols such as those found in BB might occur through alterations in stress signaling mediators such as extracellular signal regulated kinase (ERK), protein kinase C (PKC), cyclic AMP response element binding protein (CREB), and nuclear factor kappa B (NFκB). Indeed, our research has, thus far, shown that the BB protection against Aβ42
- or DA- induced decrements in intracellular calcium clearance following oxotremorine-induced depolarization in M1 muscarinic receptor (MAChR)-transfected COS-7 cells or neonatal hippocampal neurons involved reductions in phosphorylated MAPK, PKCγ, and phosphorylated CREB (17
). Similar findings, have also been seen with BB treatment in primary hippocampal cells (16
). The results showed that BB pre-treatment prevented the deficits in calcium buffering, normalized cyclic CREB and PKCγ associated with ROS signaling, and increased expression of protective ERK. Thus, these findings and those of previous studies suggest that the primary mechanisms involved in the beneficial effects of the berries may involve alterations in stress signals. However, since the concentrations utilized here may have been higher than those that occur in vivo, the effects of the treatments will have to be assessed further in whole animal models. Nevertheless, the findings here suggest that one of the mechanisms involved in the beneficial effects of BB may involve reductions in stress signals.