Modulation of multiple pathways involved in the maintenance of neuronal function during aging by fisetin
© Springer-Verlag 2009
Received: 17 July 2009
Accepted: 3 August 2009
Published: 10 September 2009
Multiple factors have been implicated in the age-related declines in brain function. Thus, it is unlikely that modulating only a single factor will be effective at slowing this decline. A better approach is to identify small molecules that have multiple biological activities relevant to the maintenance of brain function. Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also maintain mitochondrial function in the presence of oxidative stress. In addition, it has anti-inflammatory activity against microglial cells and inhibits the activity of 5-lipoxygenase, thereby reducing the production of lipid peroxides and their pro-inflammatory by-products. This wide range of actions suggests that fisetin has the ability to reduce the age-related decline in brain function.
Similar to other organs, brain function declines with age. Indeed, a decline in both cognitive and motor functions is one of the characteristics of normal aging, resulting in changes in learning and memory as well as deficits in balance and coordination. Furthermore, age is the single greatest risk factor for a variety of neurological disorders including Alzheimer’s disease (AD). Since the average age in many Western countries is increasing, identifying approaches for reducing the effects of aging on brain function is taking on a new urgency. However, in order to choose among possible approaches, it is first necessary to identify the factors that contribute to the decrease in brain function with age. Among the factors that have been proposed are alterations in redox homeostasis, gene transcription, protein modification and processing, neurotrophic factor signaling, mitochondrial function and the immune response. Given this multiplicity of factors and the strong possibility that the relative importance of these factors will vary among individuals, approaches that are directed against a single target are unlikely to be generally useful. A better approach is to identify small molecules that have multiple biological activities that can impact the multiplicity of factors that are associated with the age-related decrease in brain function.
Fisetin was originally identified in a screen for compounds that could prevent oxidative stress-induced nerve cell death . Of the ~30 flavonoids tested in this study, only two, fisetin and quercetin, were able to maintain GSH levels in the presence of oxidative stress, indicating that this is not a common property of flavonoids. Further studies showed that fisetin also possessed neurotrophic activity, promoting the differentiation of PC12 cells via activation of the Ras-ERK cascade . Again, this was a property that distinguished fisetin from almost all of the other ~30 flavonoids tested. Only quercetin, isorhamnetin and luteolin showed some differentiation-inducing activity and they were all much less effective than fisetin. Together, these observations suggested that fisetin had multiple properties that might be able to contribute to the maintenance of nerve cell function.
Unlike many of the better studied flavonoids such as quercetin and luteolin, fisetin is not particularly abundant in fruits and vegetables. The highest levels (160 μg/g) are found in strawberries  with five to tenfold lower levels in apples and persimmons. Small amounts are also found in kiwi fruit, peaches, grapes, tomatoes, onions and cucumbers . The bioavailability of fisetin from these sources has not been studied.
Maintenance of redox homeostasis by fisetin
Over the years, a number of theories have been put forth to explain the mechanisms underlying the process of aging. One of the theories that has received the most attention and research support is the free radical theory of aging (for reviews see [7, 31, 64] #1674). The current version of this theory proposes that there is an increase in the imbalance between pro-oxidants and antioxidants and, as a consequence, oxidative damage with aging which is the primary cause of the age-related declines in physiological function. The oxidants arise from several sources and include both reactive oxygen (ROS) and reactive nitrogen (RNS) species. The main sources of ROS include mitochondrial respiration, lipid peroxidation and NADPH oxidase activity. Fortunately, cells contain an arsenal of antioxidant defenses including both antioxidant enzymes and small antioxidant molecules such as the endogenous antioxidant glutathione (GSH), as well as antioxidants derived from fruits and vegetables which can normally remove the ROS/RNS generated by basic physiological functions. However, increasing age is associated with an imbalance between the production and removal of ROS/RNS resulting in oxidative stress and subsequent oxidative damage to proteins, lipids and DNA.
Although it is not entirely clear how relevant the antioxidant activity of flavonoids as measured in test tube assays is to their effects in vivo, fisetin is a relatively good antioxidant with a Trolox equivalent antioxidant capacity (TEAC) value of ~3 [36, 54]. Furthermore, fisetin was also shown to be very good at inhibiting lipid peroxidation and chelating iron . An age-related increase in iron was found in human, rat and mouse brains  and was associated with markers of oxidant-mediated damage. All of these properties could contribute to the beneficial effects of fisetin on CNS cells.
Maintenance of GSH
GSH plays a central role in maintaining cellular redox homeostasis. A fairly large number of studies have shown age-dependent decreases in total GSH and/or reduced GSH levels in the brain (for reviews see [3, 25, 53]). In addition, age-dependent increases in glutathione disulfide (GSSG), the oxidized form of GSH have been observed in both mouse  and rat  brains. This, in turn, leads to an age-dependent decrease in the GSH/GSSG ratio, suggesting a significant alteration in the redox environment of the brain with age.
The age-related decreases in total GSH that are seen in many studies could be due to increased GSH consumption, decreased GSH production or some combination of the two. Increased consumption would be consistent with an increase in ROS production with age. However, recent studies suggest that decreased production also plays an important role in the decline of brain GSH with age. These studies have all shown a good correlation between decreases in the level of glutamate cysteine ligase (GCL) activity, the rate limiting enzyme in GSH biosynthesis, and decreases in GSH levels [50, 51, 63, 71, 77]. In addition, the decreases in GCL activity are correlated with decreases in the levels of GCL protein and/or mRNA of at least one of the two subunits [50, 51]. A recent paper showed that the decrease in GCL levels, at least in liver, is due to a decrease in the level of Nrf2, the transcription factor involved in the induction of the genes encoding both chains of GCL . Moreover, the low levels of Nrf2 in the livers of 24–28-month-old rats could be restored by treatment with lipoic acid, resulting in a restoration of both GCL activity and GSH levels. This suggests that the basic transcriptional response mechanism is still present in the old rats, but that for some reason the basal set point is turned down during aging. Although further research is clearly needed to determine if decreases in Nrf2 levels are also seen in the brain with aging, these findings suggest that compounds which can increase Nrf2 might be useful for maintaining redox homeostasis in the brain and thereby helping to maintain brain function.
Over the last few years, we have developed several in vitro models that can be used to identify small molecules that are able to maintain redox homeostasis in the presence of oxidative stress. Fisetin was first identified as a potential neuroprotective compound  using one of these models, oxidative glutamate toxicity (for review see ).
More recently, we have tested the effect of fisetin against peroxynitrite toxicity. Peroxynitrite levels increase during aging and may contribute to some of the nerve cell damage associated with normal aging . In addition, peroxynitrite-mediated toxicity has been implicated in many age-related neurological disorders including ischemic stroke , AD, Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) (reviewed in ). Although peroxynitrite itself is not a free radical, it is a uniquely damaging molecule because it can initiate strong oxidation reactions through decomposition into a hydroxyl radical and nitrogen dioxide . It can also form a highly reactive nitroderivative in the presence of transition metals [6, 35] and interact directly with protein and non-protein thiol groups, leading to the depletion of cellular antioxidant defenses including GSH .
Using primary cultures of cortical neurons, in combination with the peroxynitrite generator SIN-1, we found an ~50% decrease in both intracellular GSH levels and cellular viability that could be prevented by treatment with 10 μM fisetin . Similar results with fisetin were obtained when authentic peroxynitrite was used as the toxic insult. The protection by fisetin, as well as its ability to maintain GSH levels, was inhibited by treatment with buthionine sulfoximine (BSO), an inhibitor of GCL . In contrast, BSO had no effect on the ability of glutathione monoethyl ester, a cell permeable form of GSH, to maintain GSH levels and protect the neurons from peroxynitrite toxicity. These data showed that BSO was not blocking the neuroprotective effect of fisetin through a toxic effect unrelated to GSH.
Mechanism of action
How does fisetin maintain GSH levels? In general, intracellular GSH levels are regulated by a complex series of mechanisms that include substrate availability and transport, rates of synthesis and regeneration, GSH utilization and GSH efflux to extracellular compartments (for review see ). Because glutamate and glycine occur at relatively high intracellular concentrations, cysteine is limiting for GSH synthesis in many types of cells, including nerve cells. In the extracellular environment, cysteine is readily oxidized to form cystine, so for most cell types, cystine transport mechanisms are essential to provide them with the cysteine needed for GSH synthesis. Cystine uptake in many types of cells is mediated by system X c − , a Na+-independent cystine/glutamate antiporter . System X c − is a member of the disulfide-linked heteromeric amino acid transporter family and consists of a light chain (xCT) that confers substrate specificity and a heavy chain (4F2hc) that is shared among a number of different amino acid transporters. The results with BSO suggested that fisetin increases GSH levels by either increasing cystine import and/or enhancing GCL activity.
Fisetin can maintain mitochondrial function
Age-dependent changes in mitochondrial function are of particular interest as mitochondria are thought to play a key role in the aging process, for mitochondria are both a major source of intracellular oxidants as well as a target for the damaging effects of oxidants. In mammalian cells, mitochondria are the major source of energy in the form of ATP.
Fisetin can enhance cognitive function
Normal human aging is associated with specific memory deficits including delayed recall of verbal information and declines in working memory, short-term recall, processing speed and spatial memory (for review see ). Although these deficits are distinct from those seen in neurological disorders such as AD, they can still significantly impact the quality of life. Transcriptional profiling of the human frontal cortex  and rat hippocampus  showed age-related decreases in genes involved in learning and memory. The transcription factor cAMP-response element binding protein (CREB) interacts with the cAMP-response element (CRE) in the promoter region of genes that encode proteins involved in the regulation of learning and memory (for reviews see [13, 60]). A number of studies in a wide range of animal species have shown that CREB plays a key role in the formation of long-term memory (LTM) (for review see ). CREB is a constitutively nuclear protein whose activity is regulated by phosphorylation of both subunits of the homodimer . Phosphorylation promotes the interaction of CREB with the transcriptional co-activator CREB binding protein (CBP) or its homolog p300, which stimulates the transcriptional activity of CREB. Several different kinases can phosphorylate CREB on Ser133 and positively regulate its transcriptional activity. These include protein kinase A (PKA), calmodulin dependent kinase IV, MNK1 and 2 and MSK1. The latter three kinases are all substrates of the MAP kinase ERK. Therefore, CREB activity can be regulated by the ERK signaling pathway. Using rat hippocampal slices, we demonstrated that 1 μM fisetin could induce the rapid phosphorylation of CREB and that this phosphorylation was dependent on ERK activation since inhibitors of ERK activation also blocked CREB phosphorylation .
Given these results and the known associations between CREB and learning and memory, it was next asked whether fisetin could affect long-term potentiation (LTP) in the hippocampal slices. LTP is an in vitro assay that is considered to be a good model of how memory is formed at the cellular level . Furthermore, age-related changes in cognitive function have been shown to correlate with impaired induction and maintenance of LTP . Although fisetin had no effect on basal synaptic responses in the CA1 area of rat hippocampal slices , it induced LTP in slices exposed to a weak tetanic stimulus (15 pulses at 100 Hz) that by itself failed to induce LTP. The facilitation of LTP by fisetin was dose dependent, with a maximal effect seen at 1 μM and it persisted for at least 60 min. Importantly, the facilitation of LTP by fisetin was blocked by two inhibitors of ERK activation, PD98059 and U0126. Together these data strongly support the hypothesis that ERK-dependent CREB activation by fisetin is responsible for the facilitation of LTP by fisetin. Further support for this hypothesis comes from studies with the phosphodiesterase 4 inhibitor rolipram that enhances CREB phosphorylation by preventing the breakdown of cAMP. Rolipram also had no effect on basal synaptic responses in rat hippocampal slices but facilitated LTP induced by a weak tetanic stimulus in a manner very similar to fisetin . However, in contrast to rolipram, fisetin did not increase cAMP levels in the hippocampal slices .
Among the fruits and vegetables where fisetin can be detected, the highest levels are found in strawberries . Interestingly, supplementation of the diet of 19-month-old rats with a strawberry extract for 8 weeks resulted in enhanced performance in the MWM relative to rats fed a control diet . Dietary supplementation with a strawberry extract also improved the performance of rats in the MWM in a rodent model of accelerated aging . These results support our data with fisetin, and suggest that this flavonol could be useful for reducing at least some of the learning and memory deficits that accumulate with age.
Anti-inflammatory effects of fisetin
Microglia are the resident immune cell population of the CNS, comprising 10–15% of the total cell population (for reviews see [24, 29, 74]). They play important, protective roles in the CNS such as removing pathogens and promoting tissue regeneration after injury. However, activated microglia also produce a wide array of pro-inflammatory and cytotoxic factors including cytokines, ROS, excitatory neurotransmitters and eicosanoids that can promote nerve cell damage as well as impact cognitive function. For example, the cytokine IL1-β can impair LTP . Microglia are implicated in the pathogenesis of a variety of age-associated chronic neurological disorders including AD and PD. Importantly, microglial activation is also seen in the brains of healthy, aged animals  and is thought to play a role in the exaggerated immune response that is typical of the aged brain [22, 79]. This can result in cognitive impairment and other behavioral deficits in response to stimuli that have little or no effect on these parameters in young animals. Thus, compounds that can modulate the activation of microglia in the aged brain and/or dampen their response to stimuli might have a significant benefit on brain function in the elderly.
A recent paper demonstrated that fisetin could reduce bacterial lipopolysaccharide (LPS)-induced microglial activation and neurotoxicity . Using the BV-2 microglial cell line, it was shown that fisetin was very effective at blocking LPS-induced nitric oxide production, measured as accumulation of nitrite in the culture medium, with an EC50 of ~7 μM. The same dose of fisetin also reduced LPS-induced increases in extracellular PGE2 levels as well as increases in the expression of the COX2, iNOS and interleukin-1β genes. Similar to the results obtained with other types of cells treated with fisetin and LPS [85, 92], the effects of fisetin on microglial activation appeared to be mediated by inhibition of LPS-stimulated NF-κB activation. Similar data were obtained with primary microglial cells isolated from the cerebral cortices of 1 day old mice. Importantly, these authors also showed neuroprotective effects of fisetin in a nerve/microglia co-culture system. In this assay, neuroblastoma cells were co-cultured with LPS-activated microglia with or without pre-treatment with fisetin. In the absence of fisetin, the LPS-activated microglia reduced the viability of the neuroblastoma cells by ~50%. However, following pre-treatment of the microglia with ~7 μM fisetin, the viability of the neuroblastoma cells was only reduced by ~10%. These results indicate that fisetin has anti-inflammatory activity and therefore might be effective in a variety of conditions involving the dysregulation of the immune system in the brain, including normal aging.
5-Lipoxygenase (5-LOX) metabolizes 20-carbon unsaturated fatty acids such as arachidonic acid, which are produced from membrane phospholipids by the action of phospholipases, to 5-hydroxyperoxyeicosatetraenoic acid (5-HPETE) followed by formation of the unstable intermediate leukotriene A4 (LTA4). LTA4 is then metabolized to leukotriene B4 or conjugated with GSH to form cysteinyl leukotrienes (for review see ). Via their binding to specific G-protein coupled receptors on target cells, leukotrienes can have pro-inflammatory effects. 5-LOX is expressed in the brain where its expression and activity is specifically increased in the hippocampus with age . In addition, loss of 5-LOX was associated with a reduced beta amyloid peptide burden in a transgenic mouse model of AD  suggesting that inhibition of 5-LOX might have benefits in both normal aging and AD.
Fisetin can enhance proteasome activity
The ubiquitin–proteasome pathway mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes (for reviews see [20, 43]). Several studies have shown that there is a specific decrease in proteasome activity in the hippocampus, cortex, striatum, globus pallidus and substantia nigra with aging in rodents [40, 95]. In contrast, little or no change in proteasome activity is seen in the cerebellum and brain stem. These findings are consistent with studies that have shown that proteasome activity is decreased in a variety of age-associated neurological disorders including AD, PD and ALS [8, 19, 21] and may contribute to disease progression. Interestingly, the age-related decreases in proteasome activity are generally not associated with decreased levels of overall proteasome immunoreactivity  suggesting that post-translational modifications to the proteasome are responsible for the decrease in activity. This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Indeed, microglia isolated from aged rodents show decreased proteasome activity and an impaired ability to degrade oxidized and glycated proteins .
Enhancing proteasome activity by compounds such as fisetin could prove beneficial for reducing the CNS consequences of normal aging as well as treating neurological disorders. Although the increase in proteasome activity brought about by fisetin is modest, it is similar to the increases seen with several other compounds such as dithiolethione, 3-methylcholanthrene and β-napthoflavone [44, 46]. Furthermore, dramatic increases in activity may not be compatible with the maintenance of normal cell function.
Consistent with the ability of fisetin to alter protein stability are two recent reports showing that fisetin can inhibit beta amyloid peptide fibril formation in a cell-free assay system [1, 42]. One of these studies  also showed that fisetin prevents extracellular beta amyloid peptide toxicity in the HT22 cells. Since beta amyloid peptide is thought to play a key role in the nerve cell loss that is the hallmark of AD, these results suggest that fisetin may be able to reduce the burden of beta amyloid peptide through multiple mechanisms, including inhibition of aggregation and enhancement of degradation.
Metabolism of fisetin
Flavonoids are known to be extensively metabolized following oral consumption resulting in glucuronidated, sulfated and methylated metabolites (for reviews see [59, 69]). The metabolism of fisetin was recently characterized in rats following intravenous and oral administration . Following both types of administration, the levels of free fisetin in serum declined rapidly while the levels of sulfated/glucuronidated fisetin increased. Following oral administration at 50 mg/kg, the serum concentration of fisetin sulfates/glucuronides was maintained at ~10 μM for >24 h. These results are in sharp contrast to those obtained for 5 OH and 7 OH flavone  and baicalein  where the levels of free and conjugated flavonoid never exceeded 1 μM following oral administration. When tested in an assay for antioxidant activity, the fisetin sulfates/glucuronides showed somewhat reduced but still significant activity as compared to free fisetin . This result is consistent with a recent study on the effects of glucuronidation on the ability of several different flavonoids to protect nerve and lymphoid cells from oxidative stress-induced death . Although the flavonoid glucuronides had generally higher EC50s for protection as compared to their unmodified parent, they still showed excellent activity. Furthermore, circulating flavonoid sulfates/glucuronides can be cleaved to the free form in a tissue-specific manner if there is a local release of β-glucouronidases and/or sulfatases .
Blood brain barrier penetration potential of fisetin
BBB penetration potential
This work was supported by the Michael J. Fox Foundation and the Alzheimer’s Association.
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