Derek R. Fisher

Plasmodium falciparum erythrocyte membrane protein 1 is anchored to the actin-spectrin junction and knob-associated histidine-rich protein in the erythrocyte skeleton.

A distinctive pathological feature of Plasmodium falciparum malaria is the endothelial attachment of erythrocytes infected with mature asexual-stage parasites in microvessels of the major organs. Electron-dense protrusions described as knobs are displayed on the surface of parasitized erythrocytes and act as attachment points in cytoadherence. Parasite-encoded knob-associated histidine-rich protein (KAHRP) is a major component of knobs found on the cytoplasmic side of the host cell membrane. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a family of parasite-encoded cytoadherence receptors localized to knobs on the surface of parasitized erythrocytes. Despite its high antigenic diversity, PfEMP1 has a remarkably conserved cytoplasmic domain. We demonstrate in this study that the cytoplasmic domain of PfEMP1 (VAR(CD)) binds to host spectrin and actin and to full-length KAHRP in vitro. Apparent dissociation constants determined for VAR(CD)/F-actin and VAR(CD)/KAHRP interactions are 44.9+/-6.4 and 10. 7+/-2.2 nM, respectively. Further, we provide evidence that KAHRP polypeptides self-associate in solution to form structures similar to knobs and show binding of self-associated KAHRP clusters to spectrin-actin-protein 4.1 complexes. Findings in this study suggest that PfEMP1 is localized to the knob in P. falciparum-infected erythrocytes by binding to the host spectrin-actin junction and to self-associated KAHRP through its conserved cytoplasmic domain.

Oxidative stress and inflammation in brain aging: nutritional considerations.

Aging can be defined as the condition where stressors are not counteracted by protective functions, leading to a dysregulation in development. These changes can be translated into decrements in neuronal functioning accompanied by behavioral declines, such as decreases in motor and cognitive performance, in both humans and animals. When coupled with genetic alterations, the ultimate expression of these changes is seen in diseases such as Alzheimer disease (AD). This association will be discussed in the last section of this chapter. In this review we will describe motor and cognitive deficits in behavior due to aging, and show how these deficits are related to increased vulnerability to oxidative stress, inflammation or signaling. Importantly, using muscarinic receptors as examples, we will also try to show that the sensitivity to these insults may be differentially expressed among neurotransmitter receptor subtypes.

Oxidative stress protection and vulnerability in aging: putative nutritional implications for intervention

Research indicates that vulnerability to oxidative stress (OSV) may increase in aging, suggesting that age-related neurodegenerative diseases such as Alzheimers disease (AD) or vascular dementia (VAD) may be superimposed upon a vulnerable neuronal environment. Determinations in cell models have suggested that the enhanced OSV may be the result of, (a) increases in membrane lipids, especially sphingomyelin and the sphingomyelin metabolite, sphingosine-1-phosphate, (b) decreases in glutathione, and (c) CNS distribution of OS-sensitive neuronal muscarinic receptor subtypes (e.g. M1, M2 and M4). These changes appear to enhance, (a) decrements in cellular calcium buffering following KCl-induced depolarization, and (b) cell death under OS conditions. Among the most effective agents that antagonized cellular OSV were the combination of polyphenolics found in fruits (e.g. blueberry extract) with high antioxidant activity. Subsequent experiments using dietary supplementation with fruit (strawberry) or vegetable (spinach) extracts have shown that such extracts are also effective in forestalling and reversing the deleterious effects of behavioral aging in F344 rats. Thus, it appears that the beneficial effects of the polyphenolics found in fruits and vegetables in neuronal aging and behavior may be similar to those seen with respect to carcinogenesis and cardiovascular disease.

Anthocyanin-rich açai (Euterpe oleracea Mart.) fruit pulp fractions attenuate inflammatory stress signaling in mouse brain BV-2 microglial cells.

Age-related diseases of the brain compromise memory, learning, and movement and are directly linked with increases in oxidative stress and inflammation. Previous research has shown that supplementation with berries can modulate signaling in primary hippocampal neurons or BV-2 mouse microglial cells. Because of their high polyphenolic content, fruit pulp fractions of açai ( Euterpe oleracea Mart.) were explored for their protective effect on BV-2 mouse microglial cells. Freeze-dried açai pulp was fractionated using solvents with different polarities and analyzed using HPLC for major anthocyanins and other phenolics. Fractions extracted using methanol (MEOH) and ethanol (ETOH) were particularly rich in anthocyanins such as cyanidin, delphinidin, malvidin, pelargonidin, and peonidin, whereas the fraction extracted using acetone (ACE) was rich in other phenolics such as catechin, ferulic acid, quercetin, resveratrol, and synergic and vanillic acids. Studies were conducted to investigate the mitigating effects of açai pulp extracts on lipopolysaccharide (LPS, 100 ng/mL) induced oxidative stress and inflammation; treatment of BV-2 cells with acai fractions resulted in significant (p < 0.05) decreases in nitrite production, accompanied by a reduction in inducible nitric oxide synthase (iNOS) expression. The inhibition pattern was emulated with the ferulic acid content among the fractions. The protection of microglial cells by açai pulp extracts, particularly that of MEOH, ETOH, and ACE fractions, was also accompanied by a significant concentration-dependent reduction in cyclooxygenase-2 (COX-2), p38 mitogen-activated protein kinase (p38-MAPK), tumor necrosis factor-α (TNFα), and nuclear factor κB (NF-κB). The current study offers valuable insights into the protective effects of açai pulp fractions on brain cells, which could have implications for improved cognitive and motor functions.

Cellular and Behavioral Effects of Stilbene Resveratrol Analogues : Implications for Reducing the Deleterious Effects of Aging

Research suggests that polyphenolic compounds contained in fruits and vegetables that are rich in color may have potent antioxidant and anti-inflammatory activities. The present studies determined if stilbene (e.g., resveratrol) compounds would be efficacious in reversing the deleterious effects of aging in 19 month old Fischer 344 rats. Experiment I utilized resveratrol and six resveratrol analogues and examined their efficacies in preventing dopamine-induced decrements in calcium clearance following oxotremorine-induced depolarization in COS-7 cells transfected with M1 muscarinic receptors (MAChR) that we have shown previously to be sensitive to oxidative stressors. Experiment II utilized the most efficacious analogue (pterostilbene) from experiment I and fed aged rats a diet with a low (0.004%) or a high (0.016%) concentration of pterostilbene. Results indicated that pterostilbene was effective in reversing cognitive behavioral deficits, as well as dopamine release, and working memory was correlated with pterostilbene levels in the hippocampus.

AGE-RELATED NEURODEGENERATION AND OXIDATIVE STRESS: Putative Nutritional Intervention

This review describes age-related changes that occur in neuronal function and cites evidence to show that these alterations may be the result of increased sensitivity to oxidative stress (OS). Evidence is presented to show that the abilities to mitigate the OS effects and to repair the damage from OS show decline as a function of age. Results from age- and OS-sensitive tests are given; these results indicate that one of the major sites of action of OS is the membranes, especially if compromised by high amounts of sphingomyelin, and one of the major effects of OS is to further alter the calcium disregulation in aging. It is suggested that attempts to increase antioxidant protection through diets comprised of fruits and vegetables identified as being high in total antioxidant activity might prevent or reverse the deleterious OS effects on neuronal aging.

Membrane and Receptor Modifications of Oxidative Stress Vulnerability in Aging: Nutritional Considerations

ABSTRACT: Evidence suggests that oxidative stress (OS) may contribute to the pathogenesis of age‐related decrements in neuronal function and that OS vulnerability increases as a function of age. In addition to decreased endogenous protection, increases in OS vulnerability may result from changes in membrane lipids and distribution of receptor subtype. Using a PC‐12 cell model system, we have shown that H2O2 or dopamine (DA) exposure induced deficits in the cells ability to clear (extrude/sequester, E/S) Ca2+ that are similar to those seen in aging. When plasma membrane concentrations of sphingomyelin (SPM) were used, the SPM metabolite, sphingosine‐1‐phosphate was increased to the same levels as those seen in aging, and enhancement of OS‐induced decreases in calcium E/S following KCL depolarization was observed. Differential decreases in CA2+ E/S were also seen following DA‐induced OS in COS‐7 cells transfected with one of five muscarinic receptor subtypes. Cells transfected with either M1, M2, or M4 receptors showed significantly greater vulnerability to OS (as expressed by greater decrements in calcium E/S and cell death) than those transfected with M3 or M5 receptors. The vitamin E analogue, Trolox, and the nitrone‐trapping agent, PBN, were not effective in altering E/S decrements but were effective in preventing cell death 24 h after OS exposure. These findings suggest that putative regional (e.g., striatum and hippocampus) increases in OS vulnerability and loss of neuronal function in aging may be dependent upon membrane SPM concentration and receptor subtype. In related studies, attempts were made to determine whether increased OS protection via nutritional increases in antioxidant levels in rats [using diets supplemented with vitamin E (500 IU/kg), strawberry extracts (9.4 g/kg dried aqueous extract, DAE), spinach (6.7 g/kg DAE), or blueberry extracts (10 g/kg DEA for six weeks)] would protect against exposure to 100% O2 (a model of accelerated neuronal aging). Results indicated that these diets were effective in preventing OS‐induced decrements in several parameters (e.g., nerve growth factor decreases), suggesting that although there may be increases in OS vulnerability in aging, phytochemicals present in antioxidant‐rich foods may be beneficial in reducing or retarding the functional central nervous system deficits seen in aging or oxidative insult.

Differential protection among fractionated blueberry polyphenolic families against DA-, Aβ42 and LPS-induced decrements in Ca2+ buffering in primary hippocampal cells

It has been postulated that at least part of the loss of cognitive function in aging may be the result of deficits in Ca(2+) recovery (CAR) and increased oxidative/inflammatory (OX/INF) stress signaling. However, previous research showed that aged animals supplemented with blueberry (BB) extract showed fewer deficits in CAR, as well as motor and cognitive functional deficits. A recent subsequent experiment has shown that DA- or Abeta(42)-induced deficits in CAR in primary hippocampal neuronal cells (HNC) were antagonized by BB extract, and (OX/INF) signaling was reduced. The present experiments assessed the most effective BB polyphenol fraction that could protect against OX/INF-induced deficits in CAR, ROS generation, or viability. HNCs treated with BB extract, BB fractions (e.g., proanthocyanidin, PAC), or control medium were exposed to dopamine (DA, 0.1 mM), amyloid beta (Abeta(42), 25 muM) or lipopolysaccharide (LPS, 1 microg/mL). The results indicated that the degree of protection against deficits in CAR varied as a function of the stressor and was generally greater against Abeta(42) and LPS than DA. The whole BB, anthocyanin (ANTH), and PRE-C18 fractions offered the greatest protection, whereas chlorogenic acid offered the lowest protection. Protective capabilities of the various fractions against ROS depended upon the stressor, where the BB extract and the combined PAC (high and low molecular weight) fraction offered the best protection against LPS and Abeta(42) but were less effective against DA-induced ROS. The high and low molecular weight PACs and the ANTH fractions enhanced ROS production regardless of the stressor used, and this reflected increased activation of stress signals (e.g., P38 MAPK). The viability data indicated that the whole BB and combined PAC fraction showed greater protective effects against the stressors than the more fractionated polyphenolic components. Thus, these results suggest that, except for a few instances, the lesser the polyphenolic fractionation, the greater the effects, especially with respect to prevention of ROS and stress signal generation and viability.

Fruit extracts antagonize Aβ- or DA-induced deficits in Ca2+ flux in M1-transfected COS-7 cells

Evidence suggests that there is a selective sensitivity to oxidative stress (OSS) among muscarinic receptor (MAChR) subtypes with M1, M2 and M4 showing > OSS than M3 or M5 subtypes in transfected COS-7 cells. This may be important in determining the regional specificity in neuronal aging and Alzheimer disease (AD). We assessed the effectiveness of blueberry (BB) and other high antioxidant (HA) fruit extracts (boysenberry, BY; cranberry, CB; black currant, BC; strawberry, SB; dried plums, DP; and grape, GR) on the toxic effects of Abeta 25-35 (100 microM, 24 hrs) and DA (1 mM, 4 hrs) on calcium buffering (Recovery) following oxotremorine (750 microM) -induced depolarization in M1AChR-transfected COS-7 cells, and on cell viability following DA (4 hrs) exposure. The extracts showed differential levels of Recovery protection in comparisons to the non-supplemented controls that was dependent upon whether DA or Abeta was used as the pretreatment. Interestingly, assessments of DA-induced decrements in viability revealed that all of the extracts had some protective effects. These findings suggest that the putative toxic effects of Abeta or DA might be reduced by HA fruit extracts.

Age-related toxicity of amyloid-beta associated with increased pERK and pCREB in primary hippocampal neurons: reversal by blueberry extract.

Further clarification is needed to address the paradox that memory formation, aging and neurodegeneration all involve calcium influx, oxyradical production (ROS) and activation of certain signaling pathways. In aged rats and in APP/PS-1 mice, cognitive and hippocampal Ca(2+) dysregulation was reversed by food supplementation with a high antioxidant blueberry extract. Here, we studied whether neurons were an important target of blueberry extract and whether the mechanism involved altered ROS signaling through MAP kinase and cyclic-AMP response element binding protein (CREB), pathways known to be activated in response to amyloid-beta (Aβ). Primary hippocampal neurons were isolated and cultured from embryonic, middle-age or old-age (24 months) rats. Blueberry extract was found to be equally neuroprotective against Aβ neurotoxicity at all ages. Increases in Aβ toxicity with age were associated with age-related increases in immunoreactivity of neurons to pERK and an age-independent increase in pCREB. Treatment with blueberry extract strongly inhibited these increases in parallel with neuroprotection. Simultaneous labeling for ROS and for glutathione with dichlorofluorescein and monochlorobimane showed a mechanism of action of blueberry extract to involve transient ROS generation with an increase in the redox buffer glutathione. We conclude that the increased age-related susceptibility of old-age neurons to Aβ toxicity may be due to higher levels of activation of pERK and pCREB pathways that can be protected by blueberry extract through inhibition of both these pathways through an ROS stress response. These results suggest that the beneficial effects of blueberry extract may involve transient stress signaling and ROS protection that may translate into improved cognition in aging rats and APP/PS1 mice given blueberry extract.