Preventing and Treating Neurological Disorders with the Flavonol Fisetin

Fisetin and Alzheimer’s disease and dementia

Alzheimer’s disease (AD) is the most common type of dementia. It is characterized pathologically by the presence of both extracellular neuritic plaques containing amyloid beta (Aβ) peptide and intracellular neurofibrillary tangles containing tau [17]. Clinically, AD results in a progressive loss of cognitive ability and eventually daily function activities [18, 19]. Currently approved therapies are only symptomatic, providing moderate improvements in memory without altering the progression of the disease pathology [20, 21]. Although a large number of clinical trials have been conducted in recent years with drug candidates designed to directly or indirectly reduce the amyloid plaque load, all of these trials have failed [2, 3].

Three different types of models have been used to study the possible beneficial effects of fisetin in AD: interventional, transgenic and sporadic. For the interventional studies, Aβ is injected directly into the cerebral ventricles in the brains of the mice (intracerebroventricular (icv) injection). There are numerous transgenic models of AD that are based on the mutations associated with the rare genetic form of the disease (familial AD or FAD). The models develop different degrees of cognitive impairment, levels of plaques and tangles, synaptic loss, gliosis and nerve cell death depending on the type and number of mutations [1–5] [22]. Although AD drug discovery has largely focused on these FAD models, this form of the disease accounts for only ∼ 1% of the total cases [23], and may be quite distinct from the much more prevalent, old age-associated, sporadic form of AD. Importantly, while many therapies directed against the amyloid pathway are effective in FAD transgenic mice, to date none has translated into the clinic [2]. Since old age is by far the greatest risk factor for AD [23, 24], animal models that incorporate aging into disease development may prove more useful for the development of therapies. One mouse model of aging that also develops characteristics of AD is the senescence-accelerated prone 8 (SAMP8) mouse that was developed in Japan by selective breeding of a rapidly aging phenotype. These mice exhibit a progressive, age-associated decline in brain function similar to human sporadic AD patients [reviewed in 25–27].

In all three types of AD models (icv Aβ injection, 2×FAD mice, SAMP8 mice), fisetin consistently prevented the loss of cognitive function [28–30]. Both oral administration in the food (500 mg/kg food; ∼45 mg/kg body weight (bw)/day) [28, 29] and intraperitoneal (ip) injection (20 mg/kg bw/day) [30] proved effective. In all of the models, fisetin maintained the levels of synaptic proteins and reduced markers of inflammation. It also reduced multiple markers of oxidative stress and particularly lipid peroxidation and activated the ERK pathway which is involved in both memory [31] and neurotrophic factor production and signaling [13]. However, the effects on the levels of soluble and insoluble Aβ varied between the different models suggesting that this may not be a critical target of fisetin in AD.

Although caused by dysfunction of the vascular endothelium, vascular dementia results in a similar loss of cognitive function as classical AD and is often a component of AD [32]. One approach to modeling vascular dementia is to treat rats with L-methionine which induces neurological changes similar to those seen in vascular dementia. Oral administration of fisetin (5–25 mg/kg bw/day) dose dependently reduced the impact of L-methionine treatment on cognitive function, vascular function and markers of neurodegeneration when administered to rats for 2 weeks during a 4 week treatment with L-methionine [33]. Thus, fisetin is effective at preventing loss of cognitive function and maintaining brain health in multiple, distinct models of dementia.

Fisetin and Parkinson’s disease

Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disease and the second most common neurodegenerative disease after AD. The characteristic features of PD include resting tremor, bradykinesia (slowness of movement), rigidity and postural instability (for review see 34). PD is also associated with a variety of non-motor symptoms that contribute to disability. The majority of PD cases are sporadic with only about 10% of PD patients reporting a family history of the disease [35]. As with AD, age is the greatest risk factor for disease development. The pathological hallmark of PD is the degeneration of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) [34]. Since these neurons synapse with neurons in the striatum, their loss leads to the depletion of striatal dopamine. PD is also characterized by the presence of cytoplasmic protein aggregates, called Lewy bodies, in the remaining DA neurons of the SNc. Currently available treatments only improve symptoms but do not stop disease progression. Importantly, by the time that the first symptoms appear, striatal dopamine is reduced by ∼80% and ∼60% of the DA neurons of the SNc have died [36].

Modeling of PD in animals has most often involved treatment with a toxin such as a pesticide or other toxic compound that has been associated with PD in vivo. One of the most widely used models employs the toxin MPTP while chronic, low dose administration of the pesticide rotenone is gaining in popularity [37, 38]. However, neither of these models recapitulates all of the aspects of human PD [37, 38] and both are rapid onset as compared to the slow, age-dependent development of PD in human patients.

Several years ago, we tested fisetin in the MPTP model in mice [39]. Fisetin was administered orally at 10 and 25 mg/kg bw/day beginning one day before the MPTP treatment and continuing for 7 days after the treatment. At 25 mg/kg, fisetin increased striatal dopamine levels by slightly more than 2-fold and even at 10 mg/kg there was a 70% increase in striatal dopamine levels. Tyrosine hydroxylase (TH) immunoreactivity was also examined in the striatum, the site of DA nerve terminals that originate in the SN. MPTP caused a significant loss of TH immuno reactivity and this was largely prevented by fisetin treatment.

Very recently, fisetin was tested in the chronic, low dose rotenone model in rats using oral administration of 10 and 20 mg/kg bw/day beginning 10 days after the start of the rotenone treatment and continuing until the end of the treatment [40]. Both doses of fisetin improved motor function and reduced rotenone-mediated decreases in striatal dopamine levels and TH immuno reactivity although the higher dose was more effective. Both doses also improved mitochondrial function and markers of oxidative stress in the midbrain, the area affected in PD. Together these data indicate that fisetin is efficacious in two distinct rodent models and strongly support further testing in additional PD models.

Fisetin and Huntington’s disease

Huntington’s disease is a late onset, progressive and fatal neurodegenerative disorder characterized by movement and psychiatric disturbances as well as cognitive impairment. There is, at present, no cure. HD is an autosomally dominant inherited disease that is caused by an unstable expansion of a trinucleotide repeat (CAG) that encodes an abnormally long polyglutamine tract in the huntingtin protein. The age at disease onset inversely varies with the CAG repeat number. The identification of the disease-causing mutation has allowed the development of a number of cellular and animal models of HD and these have been used to try to elucidate the mechanisms underlying disease development and progression [for reviews see 41, 42–44].

Fisetin was tested in the R6/2 mouse model of HD which is a transgenic line that has an aggressive disease phenotype and shortened lifespan [45]. Fisetin was fed to R6/2 mice and their wild type littermates in the food (500 mg/kg food; ∼45 mg/kg bw/day) beginning at ∼6 weeks of age. The mice were tested for motor function using the rotorod from ∼7–13 weeks of age and survival was followed. At the time of acquisition of the animals, rotorod performance was already impaired in the R6/2 mice as compared to their wild type littermates. Motor performance in the rotorod test declined significantly more rapidly in the R6/2 mice on the control diet as compared to those on the fisetin diet. Similarly, while the median life span of the R6/2 mice on the control diet was 104 days, that of fisetin-fed mice was increased by ∼30% to 139 days. The in vivo mechanisms underlying the effects of fisetin were not explored in this study. While encouraging, further studies are needed to support and extend these observations.

Fisetin and amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by the loss of the motor neurons that control the voluntary movement of muscles resulting in paralysis and death, usually within 5 years of a diagnosis [46]. Approximately 10% of ALS cases are due to heritable gene mutations but different gene mutations are increasingly being found in patients with no family history of ALS suggesting that the genetic component is more complicated than originally thought [47]. Although there are three FDA-approved drugs for ALS, they all have very modest effects on survival (https://alsnewstoday.com/approved-treatments/).

The most commonly used mouse model of ALS is based on Cu/Zn superoxide dismutase 1 (SOD1), the first gene mutation that was shown to cause ALS [47]. SOD1 mutations are found in ∼20% of patients with the familial form of ALS. The most extensively used of these mouse models in the SOD1-G93A transgenic mouse which is characterized by abnormal protein aggregation, motor neuron loss, axonal denervation, progressive paralysis, and reduced lifespan [47].

Oral administration of fisetin (9 mg/kg/day) beginning at 2 months of age significantly delayed the development of motor deficits, reduced their rate of progression and increased lifespan [48] in the SOD1-G93A mouse. This correlated with a significant increase in the motor neuron count in the spinal cord. At the molecular level, fisetin increased the levels of phosphorylated and therefore activated ERK as well as the antioxidant protein heme oxygenase 1. Interestingly, fisetin also increased ERK phosphorylation in a transgenic AD model [28] and in HD flies [45] suggesting that this mechanism may at least partly contribute to its beneficial effects in these different models of neurodegenerative diseases. Further studies on fisetin and ALS are clearly warranted.

Fisetin and stroke

Fisetin and traumatic brain injury

Traumatic brain injury (TBI) is caused by a bump, blow or jolt to the head or a penetrating head injury that disrupts the normal functioning of the brain [for reviews see 65, 66]. It is a growing cause of both disability and death. Similar to stroke, TBI also evolves in phases with the initial damage to axons and membranes eventually leading to an inflammatory response that causes further damage. Despite an increasing understanding of the underlying physiological changes that occur during TBI, there are still no good treatments.

There are multiple animal models of TBI including fluid percussive injury, controlled cortical impact injury, weight drop impact acceleration injury and blast injury and each has its advantages and disadvantages [66]. Fisetin was tested in the weight drop impact acceleration injury model using mice [67]. This model results in the development of injuries similar to those seen in motor vehicle or sports accidents [66]. The mice were injected ip with 25, 50 or 75 mg/kg bw fisetin 30 min after the induction of TBI and then evaluated 1, 3 and 7 days later [67]. Fisetin dose dependently improved the neurological score and motor function at 1 and 3 days and reduced brain edema, blood brain barrier disruption and lesion volume with 50 mg/kg bw showing the best effect. These beneficial physiological effects correlated with decreases in markers of oxidative stress and apoptosis and increases in the levels of nuclear Nrf2 as well as Nrf2 target proteins in the brains. To further evaluate the role of Nrf2 in the protective effects of fisetin against TBI, the same experiment was carried out in Nrf2 knockout mice. Interestingly, while fisetin failed to reduce markers of oxidative stress in the mice lacking Nrf2, it was still able to improve the neurological score and reduce the lesion volume as well as the markers of apoptosis. Thus, other targets of fisetin appear to be critical for many of its beneficial effects against TBI. However, given these promising results, further exploration of fisetin in additional models of TBI is clearly warranted.

Fisetin, memory and age-related changes in the brain

Similar to other organs, brain function declines with age. 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 68]. Although old age is not considered a disease, a decline in brain function can significantly impact the quality of life. In addition, age-related changes in brain function contribute to the development and progression of many of the neurological diseases/disorders described above.

In our first animal study with fisetin, we tested its effects on memory in young adult mice (3 month old male C57BL/6J) using the object recognition test [69]. In this test, during the training period, mice are presented with two identical objects, which they explore for a fixed time period. To test for long term memory (LTM), the mice are presented one day later with two different objects, one of which was presented previously during the training and is thus familiar to the mice; the other object is new to them. The better the mice remember the familiar object, the more time they will spend exploring the novel object. To test the effects of fisetin in this memory task, it was administered orally (5–25 mg kg bw) to the mice before the start of the training period [70]. Rolipram, administered by injection, was used as a positive control. In a dose dependent manner, fisetin significantly increased the time the mice spent exploring the novel object indicating a significant effect on LTM.

In order to understand the physiological mechanism underlying this effect of fisetin, we asked whether fisetin could affect long-term potentiation (LTP) in hippocampal slices from rat brains. LTP is an in vitro assay that is considered to be a good model of how memory is formed at the cellular level [71]. Furthermore, age-related changes in cognitive function have been shown to correlate with impaired induction and maintenance of LTP [68]. Although fisetin had no effect on basal synaptic responses in the CA1 area of rat hippocampal slices [70], 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 the induction of LTP by fisetin was dose dependent, with a maximal effect seen at 1μM and it persisted for at least 60 min.

Importantly, these results with hippocampal slices have recently been extended to in vivo studies in anesthetized rats [72]. Similar to the experiments with the slices, oral administration of fisetin (10 & 25 mg/kg bw) dose dependently facilitated the induction of LTP in vivo. In contrast, structurally related flavonoids including quercetin, myricetin and luteolin, did not have a significant effect on the facilitation of LTP when tested at the same doses [72].

More recently, the effects of fisetin on age-related changes in the brain were tested in 24 month old rats [73]. The animals were treated daily for 6 weeks with 15 mg/kg bw fisetin dissolved in 10% DMSO and administered orally. Fisetin reduced brain markers of oxidative stress, including lipid peroxidation, and increased the levels of several antioxidant enzymes relative to vehicle-treated old rats.

Consistent with these results, mice fed fisetin (500 mg/kg food) from 85 weeks of age until death showed significantly lower levels of age-related indicators of brain pathology as compared with mice fed a control diet [74] and this correlated with a significant increase in lifespan. Thus, fisetin may not only reduce the impact of neurological diseases/disorders themselves but also improve the overall environment in the brain so that these diseases/disorders are less likely to develop.

Inhibition of oxytosis/ferroptosis

This type of regulated cell death was first described as being a form of oxidative stress-induced cell death that was initiated by the depletion of the major intracellular antioxidant glutathione (GSH) [10]. A reduction in GSH is seen in the aging brain and is accelerated in many neurological disorders/diseases [78]. Importantly, GSH loss in the brain is associated with impairments in cognitive function [78–80]. The depletion of GSH from cells leads to mitochondrial reactive oxygen species production, lipoxygenase activation, lipid peroxidation and calcium influx which initiates a form of cell death with features of both apoptosis and necrosis called oxytosis [10]. All of these changes are implicated in the nerve cell damage and death seen in neurological diseases [81]. Oxytosis appears to be very similar, if not identical, to another, recently described, form of cell death called ferroptosis [11]. We have found that most, if not all, compounds that inhibit oxytosis also inhibit ferroptosis [12] while the ferroptosis inhibitor ferrostatin-1 can also inhibit oxytosis [82]. This is of relevance because oxytosis/ferroptosis has been implicated in a number of pathological processes including neurodegenerative diseases [10, 12, 83, 84] and ICH [85]. Importantly, oxytosis/ferroptosis appears to be a process that can manifest itself over a lengthy time period before cells die, thereby offering a window for therapeutic intervention. Fisetin was first identified as a compound that could inhibit oxytosis in a nerve cell line [9] both through its ability to maintain GSH levels under conditions of stress and its ability to directly quench reactive oxygen species. Thus, at least some of the beneficial effects of fisetin seen in the context of neurological diseases are likely to be via its inhibition of oxytosis/ferroptosis.

Modulation of the gut microbiome

The gut microbiome has come under increasing attention as a potential contributor to multiple human diseases including some neurological disorders [86]. There is strong evidence that the gut microbiota is important for normal brain development and the maintenance of brain function. Thus, it is perhaps not surprising that animal and a few human studies have provided data suggesting that dysregulation of the gut microbiota contributes to PD, AD and the susceptibility to stroke and TBI [86]. However, since it is still not clear exactly what constitutes a healthy gut microbiota, how it can be altered to improve outcomes will require a great deal more study.

Most studies on the gut microbiome use DNA analysis of fecal material. However, evidence for changes in the gut microbiota can also be obtained from plasma metabolomic studies as some metabolites found in the plasma can only be made by bacteria [87, 88]. We analyzed the effects of fisetin on the plasma metabolome of 10 month old age-accelerated SAMP8 mice, an age where the mice show significant levels of cognitive dysfunction, following 7 months of fisetin treatment in the diet (500 mg/kg diet) [29]. Although fisetin only altered 11 metabolites relative to untreated 10 month old SAMP8 mice, most of these changes resulted in a prevention of the changes that occurred with aging in these mice [29]. Interestingly, 27% of these metabolites (4-hydroxyphenylacetate, indoleacetate and taurourso deoxycholate) are mainly derived from the gut microbiome [87, 88] suggesting that some of the beneficial effects of fisetin may be mediated by alterations to the gut microbiota.

More direct evidence for an effect of fisetin on the gut microbiome was obtained in a recent study on fisetin in a model of premature ovarian failure (POF) in mice [89]. POF was induced chemically over 4 weeks in the absence or presence of fisetin (100 ng/kg bw, ip). Analysis of fecal samples at the end of the study found that fisetin significantly altered the distribution and diversity of the gut microbiome in POF mice as well as the metabolic pathways associated with the microbiome. These results correlated with a significant improvement in markers of POF and a reduction in inflammatory markers in the blood. Clearly, the effects of fisetin on the microbiome is an area of research that deserves further attention.

Senolytic activity

Cellular senescence is caused by the stable arrest of the cell cycle and results in an altered cellular phenotype (for reviews see 90, 91, 92). Acute senescence plays an important role during development and in tissue repair while chronic senescence is associated not only with a failure of the arrested cell to perform its normal functions but also an increase in both local and systemic inflammation. Chronically senescent cells normally increase during aging and even higher numbers have been found in a large number of age-related diseases [90, 91] including some neurological disorders such as AD, PD and ALS [92]. Studies have shown that the removal of senescent cells can be beneficial in the context of both aging and disease [92–94]. Compounds that can selectively eliminate senescent cells are termed senolytics [91] and there is evidence that fisetin is one of these compounds.

A study published several years ago [95] showed that fisetin was much more effective at killing senescent as compared to proliferating human umbilical vein endothelial (HUVEC) cells. In contrast, fisetin was equally toxic to senescent and proliferating human preadipocytes.

These studies were subsequently extended to other cell lines as well as mouse models of senescence [74]. Using a progeroid mouse model carrying a luciferase tagged version of p16^INK4a, a marker for senescent cells, fisetin treatment in food (500 mg/kg diet) for 10 weeks beginning at 10 weeks of age not only reduced p16^INK4a expression but also other markers of senescence as well as markers of inflammation and oxidative stress in multiple tissues. Interestingly, fisetin did not need to be continuously fed to the animals to suppress markers of senescence consistent with a senolytic mode of action which would result in the elimination of senescent cells.