Neuroprotective mechanisms of red clover and soy isoflavones in Parkinson’s disease models

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by nigrostriatal degeneration and the spreading of aggregated forms of the presynaptic protein α-synuclein (aSyn) throughout the brain. PD patients are currently only treated with symptomatic therapies, and strategies to slow or stop the progressive neurodegeneration underlying the disease’s motor and cognitive symptoms are greatly needed. The time between the first neurobiochemical alterations and the initial presentation of symptoms is thought to span several years, and early neuroprotective dietary interventions could delay the disease onset or slow PD progression. In this study, we characterized the neuroprotective effects of isoflavones, a class of dietary polyphenols found in soy products and in the medicinal plant red clover (Trifolium pratense). We found that isoflavone-rich extracts and individual isoflavones rescued the loss of dopaminergic neurons and the shortening of neurites in primary mesencephalic cultures exposed to two PD-related insults, the environmental toxin rotenone and an adenovirus encoding the A53T aSyn mutant. The extracts and individual isoflavones also activated the Nrf2-mediated antioxidant response in astrocytes via a mechanism involving inhibition of the ubiquitin-proteasome system, and they alleviated deficits in mitochondrial respiration. Furthermore, an isoflavone-enriched soy extract reduced motor dysfunction exhibited by rats lesioned with the PD-related neurotoxin 6-OHDA. These findings suggest that plant-derived isoflavones could serve as dietary supplements to delay PD onset in at-risk individuals and mitigate neurodegeneration in the brains of patients. Graphical Abstract The isoflavone-rich extracts red clover and soy and the individual isoflavones daidzein and equol protect neuronal cultures against environmental and genetic triggers of Parkinson’s disease, and rescue motor deficits in rats exposed to the neurotoxin 6-OHDA.


Introduction
Parkinson's disease (PD) is a neurodegenerative disorder that affects 5% of the global population over the age of 85 1-3 . The disease involves a loss of dopaminergic neurons from the substantia nigra in the midbrain, and this neuronal loss is largely responsible for motor symptoms such as the inability to initiate movement, resting tremor, and reduced balance 4 . Pathological phenomena underlying neurodegeneration in PD include oxidative stress 5,6 , loss of mitochondrial function [7][8][9] , aggregation of the presynaptic protein α-synuclein (aSyn) [10][11][12] , and microglial activation 13 . Familial PD mutations in the SNCA gene encoding aSyn are thought to promote the formation of toxic aSyn oligomers by increasing the protein's expression levels or intrinsic aggregation propensity in the case of duplication/triplication mutations or substitution mutations, respectively 12,[14][15][16][17][18] .
Epidemiological evidence suggests that exposure to environmental toxins such as rotenone and paraquat leads to an increase in PD risk [19][20][21][22] , potentially via a mechanism involving the production in neurons of reactive oxygen species (ROS) that in turn lead to a build-up of oxidatively modified forms of aSyn with a high propensity to form potentially toxic aggregates 12,23,24 . Current PD treatments consist primarily of dopamine replacement therapy (DRT), including administration of the dopamine metabolic precursor L-DOPA or synthetic dopamine receptor agonists, but these agents only alleviate symptoms without delaying the underlying neuronal death 25 . Patients on long-term DRT eventually experience pronounced side effects that include L-DOPA-induced dyskinesias, aggression, and insomnia 26 . Accordingly, there is a critical need to identify disease-modifying therapies that slow PD progression.
Multiple lines of evidence suggest that isoflavone-rich extracts such as soy-derived products and the medicinal plant red clover (Trifolium pratense) could have neuroprotective effects in the brains of PD patients. In an ethnopharmacological survey of Pikuni-Blackfeet traditional medicine, we identified red clover as a traditional treatment for symptoms related to PD 27 . Moreover, epidemiological studies revealed that estrogen replacement therapy may be linked to improved cognitive function and delayed onset of PD and Alzheimer's disease (AD) [28][29][30][31] , in turn implying that the consumption of phytoestrogens in isoflavone-rich foods and plant medicines could slow functional decline of the aging brain. In support of this idea, isoflavones and isoflavone-rich extracts were found to exhibit neuroprotective activity in preclinical models of AD and cognitive impairment 32,33 . Individual isoflavones such as genistein, a major isoflavone found in soy, have also been reported to rescue the PD-like phenotypes of rodents exposed to MPTP or 6-OHDA by restoring motor functions and preserving dopaminergic neurons [34][35][36][37] . Although these observations suggest that isoflavones can alleviate neurodegeneration in PD models, the effects of isoflavones or isoflavone-rich botanical extracts on toxicity elicited by insults linked epidemiologically or genetically to PD are poorly understood. Polyphenols (including isoflavones) are well known for their ability to scavenge ROS 38,39 . However, the fact that brain levels of polyphenols are lower than those of endogenous glutathione suggests that polyphenols may mitigate neurotoxicity via additional protective mechanisms, including the activation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates the expression of genes involved in the cellular antioxidant response, as well as modulation of the ubiquitin-proteasome system (UPS) and rescue of mitochondrial functional deficits 27,40 .
This research study was aimed at characterizing isoflavone-rich extracts and individual isoflavones in terms of (i) their neuroprotective effects in cellular models relevant to PD; and (ii) their mechanisms of action, with an emphasis on their ability to stimulate the Nrf2-mediated antioxidant pathway, the UPS, and mitochondrial function. Our findings support the idea that medicinal plants enriched in isoflavones such as red clover and soy could form the basis of dietary interventions for individuals at risk for PD or therapeutic preparations to slow PD progression.

Materials
Chemicals were obtained from Sigma Chemical Co.

Preparation and dissolution of botanical extracts
Red clover flowers were harvested, immediately dried at 37 °C with a food processor, and the water extract was prepared as described previously to reproduce the traditional methods of preparation of red clover-based herbal remedies 27,40 . The soy extract was prepared as described from Tofu soybeans 43,44 . Prior to each experiment, extracts were dissolved in sterile deionized water (red clover) or ethanol (soy and Novasoy 400). HPLC-TOF-MS analyses were conducted on an Agilent 6220a TOF-MS, equipped with a   At least 12 images with a total of ~500 to 1000 MAP2 + neurons were analyzed per experiment for each treatment, and the data were expressed as the percentage of MAP2 + neurons that were also TH + to normalize for variations in cell plating density. Each experiment was performed using at least 3 independent preparations of embryonic cultures.

Preparation of rat primary cultures
Neurite length measurements were carried out on images taken with an automated Cytation 3 Cell Imaging Reader equipped with a 4X objective (generally, these images were the same as those used to assess dopaminergic cell viability as outlined above). Lengths of MAP2 + processes extending from TH + /MAP2 + neurons with an intact cell body (~90 neurons per sample) were measured in a blinded manner using the manual length measurement tool of the NIS Elements software (Nikon Instruments, Melville, NY). Levels of target gene cDNA (encoding HO1, GCLC, or Nrf2) were normalized to levels of control gene cDNA (encoding GAPDH), and the fold change for samples treated with a red clover extract relative to control samples was calculated using the following formula:

Treatment with ARE-EGFP reporter adenovirus
Where Ct,TG represents the crossing threshold for the target gene, Ct,CG represents the crossing threshold for the control gene, 'RC' refers to mRNA obtained from cells incubated in the presence of red clover extract, and 'control' refers to mRNA obtained from cells incubated in the absence of extract.

UPS reporter assay
UPS function was monitored as described 40  Quantification of GFP fluorescence was carried out as described previously 40 . Briefly, ROIs were generated by the GEN5 2.05 software based on the cellular size range (20 to 400 µm) and a designated fluorescence intensity threshold. For each experiment, the threshold was adjusted so that a 6-to 7-fold increase in the number of ROIs above the threshold was observed for the MG132treated culture compared to the negative-control culture. Hoechst fluorescence was quantified by generating ROIs with a fluorescence intensity threshold as described in section 2.7. Finally, the number of ROIs for GFP was divided by the total cell number (ROIs obtained from Hoechst fluorescence) for each treatment and normalized to the control value to obtain a fold-change value.

Oxygen consumption assay
Oxygen consumption was monitored as described previously 40,53 . In other experiments, isoflavone-rich extracts were tested for the ability to displace rotenone from its binding site in complex I of the electron transport chain using a competition assay.
Galactose-conditioned SH-SY5Y cells were resuspended in O 2 consumption buffer containing rotenone (50 nM) with or without red clover extract (1 µg/mL) or soy extract (1 µg/mL). Control cells were resuspended in O 2 consumption buffer without rotenone or extract. Mean O 2 consumption rates were determined from 3 independent experiments and normalized to control values as described above. Rat motor function was assessed using the open field test 1 d before the injections and at different times from 12 h after surgery to the end of the study 55,56 . The animals were placed in a Plexiglas activity box (100 × 100 × 20 cm 3 ) with food over the center. Eight infrared beams were set in an X-Y matrix, and the position of the rat relative to the beams was monitored at 200 ms intervals with in-house software. The data were used to calculate the percentage of the total box area covered.

Study design
The central hypothesis of this study was that botanical extracts rich in isoflavones can alleviate neurotoxicity elicited by insults linked epidemiologically or genetically to PD. To address this hypothesis, we characterized two extracts with high levels of isoflavones -a red clover extract 57 and a soy extract prepared from Tofu soybeans 43,44 -in terms of their ability to interfere with dopaminergic cell death in primary midbrain cultures exposed to two PD stresses: (i) rotenone, an environmental toxicant epidemiologically linked to increased PD risk 22 ; and (ii) virus encoding A53T aSyn, a familial mutant form of aSyn that exhibits accelerated fibrillization and enhanced neurotoxicity compared to the wild-type protein 15,58,59 . Moreover, we characterized an isoflavone constituent of the soy extract, daidzein, and a second isoflavone, equol (produced from daidzein by the intestinal microbiota), to determine their effects on dopaminergic neuron survival in primary midbrain cultures exposed to rotenone or A53T Ad. The extracts and individual isoflavones were also examined for their effects on Nrf2 signaling and the UPS, as well as their ability to enhance mitochondrial respiration.

Effects of a red clover extract on dopaminergic cell death elicited by PDrelated insults in primary midbrain cultures
Our first objective was to measure the neuroprotective activity of a red clover extract in a rat primary midbrain culture model. Our rationale for using this model was that these cultures consist of post-mitotic dopaminergic and GABAergic neurons and glial cells (e.g. astrocytes and microglia), and thus they reproduce key features of the midbrain region affected in PD patients 47,60 . The red clover extract was prepared from dried flowers, as described previously by Pikuni-Blackfeet traditional healers 27 , and chemical analysis of the extract via HPLC coupled with UV and MS detection revealed an abundance of isoflavones, including formononetin, biochanin A, and pratensin glycosides, and confirmed the isoflavone-rich nature of the red clover extract (Table 1).
In one set of experiments, primary midbrain cultures were exposed to rotenone in the absence or presence of the red clover extract. The cultures were co-stained for MAP2, a general neuronal marker, and TH, a selective marker of dopaminergic neurons (Fig. 1A), and scored for relative dopaminergic neuron viability by determining the ratio of TH + neurons to total MAP2 + neurons 15,27,40,47 . Cultures exposed to rotenone alone exhibited a ~20-30% decrease in relative dopaminergic cell viability, and this decrease was abrogated in cultures exposed to rotenone plus red clover extract (Fig. 1B). Moreover, neurite length measurements revealed a decrease in the lengths of processes extending from MAP2 + /TH + neurons in cultures exposed to rotenone compared to control cultures, consistent with the 'dying back' degeneration of dopaminergic neurites that is thought to precede cell death observed in the brains of PD patients 61,62 , and this decrease in neurite lengths was alleviated in cultures treated with rotenone in the presence of red clover extract (Fig. 1C). These results suggested that polyphenols in the red clover extract interfered with dopaminergic cell death and neurite retraction elicited by rotenone.
In a second set of experiments, primary midbrain cultures were transduced with A53T Ad, previously shown to be selectively toxic to MAP2 + /TH + neurons 15 , in the absence or presence of red clover extract. The cultures were co-stained for TH and MAP2 and analyzed for relative dopaminergic cell survival as outlined above. Cultures treated with virus plus extract had a higher percentage of TH + neurons compared to cultures treated with virus alone (Fig. 1D), suggesting that red clover polyphenols inhibited aSyn-mediated dopaminergic neurotoxicity.

Effects of a red clover extract on astrocytic Nrf2/ARE signaling
The transcription factor Nrf2 is a master regulator of the cellular antioxidant response 63 . In the cytoplasm, Nrf2 is sequestered by Kelch-like ECH-associated protein 1 (Keap1) 64 and targeted for degradation by the UPS 65 . Under conditions of oxidative stress, the interaction between Nrf2 and Keap1 is disrupted, resulting in Nrf2 translocation to the nucleus where it binds AREs in the regulatory region of its target genes 66,67 . The Nrf2-mediated response is primarily activated in astrocytes, enabling their production and secretion of glutathione metabolites that are subsequently taken up by neighboring neurons 68,69 .
Evidence suggests that rotenone and aSyn elicit neurotoxicity at least in part by triggering an increase in oxidative stress 9,70,71 . Thus, we hypothesized that the red clover extract could protect against neurotoxicity elicited by rotenone or A53T aSyn by activating the Nrf2-mediated antioxidant response 72 . To address this hypothesis, primary cortical astrocytes were transduced with a reporter adenovirus encoding EGFP downstream of DNA regulatory sequences encompassing two antioxidant response elements (AREs) and a minimal promoter derived from the mouse HO-1 gene 27,40,41,49,50 . The transduced cultures were incubated in the absence or presence of red clover extract for 24 h and examined for levels of EGFP fluorescence ( Fig. 2A). Astrocytes treated with extract exhibited greater fluorescence compared to untreated cells, suggesting that red clover polyphenols activated the Nrf2/ARE antioxidant response ( Fig. 2B-C). We observed a similar effect in mixed primary cortical cultures consisting of neurons, astrocytes, and microglia, suggesting that the presence of neurons and other glial cell types did not affect the ability of the red clover extract to activate Nrf2 transcriptional activity in astrocytes (Fig. 2D). To confirm that the observed increase in EGFP fluorescence reflected an increase in Nrf2-mediated transcription, we quantified mRNAs encoding Nrf2 target genes by qRT-PCR. In agreement with the results obtained from the EGFP reporter assay, we found that astrocytes treated with red clover extract exhibited an up-regulation of mRNA encoding HO1 or GCLC compared to control cells (Fig. 2E, F), and the observed timing of expression of these two Nrf2-regulated genes was consistent with previous findings 73 .
In subsequent experiments, we assessed whether the red clover extract could activate the Nrf2/ARE pathway in the same cellular context as that in which the extract conferred neuroprotection against toxicity elicited by rotenone or A53T aSyn. To address this question, primary midbrain cultures or astrocytes isolated from these cultures were transduced with the ARE-EGFP reporter adenovirus and incubated in the absence or presence of extract. Surprisingly, isolated astrocytes and neuron-glia cultures from embryonic rat midbrain exhibited similar levels of EGFP fluorescence after incubation with or without extract ( Supplementary Fig. 1A, B). In summary, our data indicate that the ability of a red clover extract to activate Nrf2 signaling in cultured astrocytes depends on the brain region from which they were derived (e.g. cortex or midbrain), and mechanisms of Nrf2 activation mediated by the red clover extract and reflected by our ARE-EGFP reporter may not be operative in midbrain cultures.

Mechanism of red clover-mediated Nrf2 activation in cortical astrocytes
Polyphenols have been reported to up-regulate Nrf2 transcriptional activity via various mechanisms. For example, they can trigger Keap1 modification, either by reacting directly with Keap1 regulatory cysteine residues or indirectly by promoting ROS-mediated Keap1 oxidation [74][75][76][77][78] .
In turn, Keap1 modification results in disruption of the Nrf2-Keap1 interaction, Nrf2 stabilization, and an increase in Nrf2 nuclear translocation and transcriptional activity. Some polyphenols interfere with the UPS [79][80][81][82][83] , and a loss of UPS function has been linked to the activation of Nrf2 and an increase in the expression of its target genes [84][85][86] . Other polyphenols, including the flavonoid curcumin, have also been shown to induce increased expression of the Nrf2 gene [87][88][89] . In the next phase of our study, we carried out experiments aimed at elucidating which of these mechanisms could be involved in the activation of Nrf2 signaling in cortical astrocytes by the red clover extract.
To determine whether a pro-oxidant effect was necessary for Nrf2 activation by the red clover extract, we tested the effect of the extract on Nrf2 transcriptional activity in primary cortical astrocytes cultured in the absence or presence of N-acetyl cysteine (NAC), a cell-permeable antioxidant molecule that serves as a ROS scavenger and a glutathione precursor 48,60 . Astrocytes transduced with the ARE/EGFP reporter virus and treated with red clover extract plus NAC exhibited similar fluorescence levels compared to transduced astrocytes treated with extract alone (Fig. 3), suggesting that the activation of Nrf2 transcriptional activity by the extract was not affected by treatment with NAC, and, therefore, occurred via a ROS-independent mechanism 48, 60 .
To address whether Nrf2 activation by the red clover extract could involve UPS inhibition, we tested the effect of the extract on levels of GFP fluorescence in primary cortical astrocytes transduced with a reporter adenovirus encoding GFPu, an unstable form of GFP that is linked to the CL1 degron 40,42,51,52 . Under physiological conditions, GFPu is efficiently degraded, whereas inhibition of the UPS results in accumulation of GFPu and increased cellular fluorescence. GFPuexpressing astrocytes exhibited greater fluorescence when cultured in the presence versus the absence of the red clover extract (Fig. 4A), suggesting that polyphenols in the extract inhibit UPS activity. As a control, we confirmed that UPS inhibition led to the activation of Nrf2 signaling in our cortical astrocyte model by showing that astrocytes transduced with the ARE-EGFP reporter virus showed an increase in EGFP fluorescence when cultured in the presence of the proteasome inhibitor MG132 (Fig. 4B). Curcumin, a polyphenol previously reported to interfere with UPS function 90 , induced an increase in GFP fluorescence in GFPu-expressing astrocytes (Supplementary Fig.   2A) and potently activated the Nrf2 pathway in rat cortical astrocytes, rat mixed cortical or midbrain cultures, and human iPSC-derived astrocytes ( Supplementary Fig. 2B), further supporting a link between UPS inhibition and Nrf2 activation.
To assess whether Nrf2 activation by the red clover extract could involve an increase in the expression of the Nrf2 gene, we quantified Nrf2 mRNA in cortical astrocytes via qRT-PCR. The results indicated that Nrf2 mRNA levels were increased by ~15% in cells incubated with extract for 24 h compared to untreated cells, with a similar trend evident in cells treated with extract for 6 h ( Supplementary Fig. 3).
Collectively, these data suggest that polyphenols in the red clover extract inhibit UPS activity and induce a modest increase in Nrf2 mRNA levels in cortical astrocytes. We infer that both of these effects could account at least in part for the ability of the red clover extract to activate Nrf2 signaling in these cells.

Neuroprotective effects of a soy extract and individual isoflavones
Our next objective was to examine the neuroprotective activities of an isoflavone-rich soy extract and two individual isoflavones, daidzein and equol (Fig. 5A), against toxicity elicited by PD-related insults in primary midbrain cultures. Previous HPLC analyses of the phytochemical composition of soy extracts have revealed an enrichment in isoflavones including genistein and daidzein (genistein/daidzein ratio consistently around 1.4), and lower levels of glycitein 43,44 . Our rationale for characterizing effects of daidzein is that it is an important constituent of soy products, and equol was examined because it is a metabolic product of daidzein in the intestine (i.e. it has translational significance) and exhibits greater antioxidant activity than daidzein itself [91][92][93] . Cultures treated with rotenone or A53T Ad plus soy extract showed a strong trend towards greater dopaminergic cell viability compared to cultures treated with rotenone or A53T virus alone (Fig. 5B,F). Furthermore, rotenone neurotoxicity was alleviated in midbrain cultures treated with daidzein (100 nM, with a trend towards a neuroprotective effect at 50 nM) or equol (50 nM) (Fig. 5C-E). An increase in dopaminergic neuron viability was also observed in A53T-expressing cultures treated with daidzein at a concentration as low as 25 nM (Fig. 5G). These data suggest that soy polyphenols and the isoflavones daidzein and equol interfere with neurotoxicity elicited by rotenone and A53T aSyn.
In the next set of experiments, we examined whether the soy extract, daidzein, or equol could activate the astrocytic Nrf2/ARE antioxidant response. To address this question, primary cortical astrocytes were transduced with the ARE/EGFP reporter adenovirus and incubated in the absence or presence of the extract or individual isoflavones. Quantification of EGFP fluorescence revealed that none of the treatments induced an increase in the EGFP signal ( Supplementary Fig.   4). However, in contrast to a red clover extract, the soy extract activated the Nrf2 response in human iPSC-derived astrocytes (Fig. 6). Additional analyses revealed that cortical astrocytes transduced with the GFPu reporter virus exhibited a modest increase in GFP fluorescence when treated with the soy extract (at a relatively high concentration of 20 μg/mL) or equol, but no increase in fluorescence upon incubation with daidzein ( Supplementary Fig. 5). These data suggest that (i) the neuroprotection elicited by soy polyphenols, daidzein, and equol may not rely on activation of Nrf2 signaling in cortical astrocytes, whereas a protective antioxidant activity may be induced in other subtypes of astrocytes; and (ii) soy polyphenols and equol, but not daidzein, interfere with UPS function in primary cortical astrocytes.

Effects of red clover and soy extracts on rotenone-induced impairment of mitochondrial respiration
Several lines of evidence suggest that neurotoxicity elicited by rotenone and aSyn results from various pathogenic events including disruption of mitochondrial respiration and oxidative damage 9,71,[94][95][96] . Accordingly, we hypothesized that the red clover and soy extracts might protect dopaminergic neurons against toxicity elicited by rotenone or aSyn by alleviating mitochondrial dysfunction. To address this hypothesis, we developed an assay aimed at measuring O 2 consumption in human SH-SY5Y neuroblastoma cells challenged with rotenone in the absence or presence of extract prior to performing the assay. To ensure that the cells relied on oxidative phosphorylation as a primary source of energy production, the cultures were maintained in media containing galactose instead of glucose 40, 97, 98 . Cells exposed to rotenone exhibited a ~40% decrease in the rate of O 2 consumption compared to untreated cells, and this decrease was largely reversed in cells pre-treated with extracts prepared from red clover (1 µg/mL) or soy (1 µg/mL), or with daidzein (50 nM) (Fig. 7A). These results suggested that the two extracts and daidzein interfered with rotenone-induced mitochondrial functional deficits.
A number of polyphenols have been reported to compete with rotenone for ubiquinone binding sites in complex I of the electron transport chain 99 . Thus, we hypothesized that the red clover and soy extracts stimulate mitochondrial respiration by displacing rotenone from its binding site in complex I. To address this hypothesis, we developed a competition assay aimed at monitoring the effects of polyphenols on rotenone-mediated deficits in mitochondrial O 2 consumption. Galactoseconditioned SH-SY5Y cells incubated in the presence of rotenone in the O 2 consumption assay buffer exhibited a decrease in the rate of O 2 consumption compared to untreated cells, and this inhibitory effect was partially rescued upon adding the red clover extract, but not the soy extract ( Fig. 7B,C). These results suggest that the red clover extract, but not the soy extract, preserves mitochondrial function at least in part by displacing rotenone from its binding site in complex I.

Effects of a soy extract on 6-OHDA-induced motor dysfunction
To investigate the potential neuroprotective activity of soy isoflavones in an in vivo PD model, we tested a soy extract for the ability to alleviate motor deficits in 6-OHDA-treated rats, a classic neurotoxin model of PD. Unilateral, stereotaxic injection of 6-OHDA in the MFB or striatum results in a progressive phenotype involving dopaminergic cell death and depletion of striatal dopamine on the lesioned side [100][101][102] . The resulting imbalance in striatal dopamine causes pronounced motor deficits. For these experiments, we used another soy extract, Novasoy 400, which is enriched in genistin, daidzein, and glycitin. Rats received daily ip injections of extract or control vehicle prior to and after lesioning with 6-OHDA in the right MFB (Fig. 8A). Strikingly, rats injected with soy extract exhibited less pronounced motor defects in the open field test compared to rats treated with vehicle ( Fig. 8B). Two-way ANOVA revealed a significant effect of time (p≤0.0001), treatment (p≤0.0001), and time x treatment (p≤0.0001). Lesioned animals treated with soy extract showed an increase in the percentage area covered at each time point compared to lesioned animals treated with vehicle, with a maximum increase of ~2-fold on day 10 (i.e. 9 days after 6-OHDA infusion). These results indicate that soy polyphenols alleviate motor dysfunction exhibited by rats exposed to 6-OHDA, potentially by interfering with nigrostriatal degeneration in these animals.

Isoflavone-rich extracts and individual isoflavones protect against neurotoxicity elicited by rotenone and A53T aSyn.
The objective of this study was to examine the neuroprotective capacity and mechanisms of action of two isoflavone-rich extracts, prepared from red clover and soy, and the individual isoflavones daidzein and equol, a daidzein metabolite. We found that all of the extracts and isoflavones tested alleviated (or showed a trend towards alleviating) rotenone-and A53T-induced neurotoxicity in a primary midbrain culture model of PD (Figs. 1 and 5). These findings are consistent with previous data showing a rescue of dopaminergic cell loss in 6-OHDA-lesioned rats supplemented with a red clover diet 103 , and the ability of individual isoflavones to attenuate motor and non-motor symptoms induced by exposure to the toxins MPTP and 6-OHDA 34,35 . Because the red clover and soy extracts examined in this study consist of a complex mixture of isoflavones, we infer that synergistic interactions involving multiple isoflavones could potentially play a role in the extracts' neuroprotective activities [104][105][106] . At the same time, our observation that daidzein and equol interfered with toxicity elicited by rotenone or A53T aSyn indicates that individual isoflavones can also achieve neuroprotective effects in PD models. Interestingly, equol is a metabolite of daidzein produced by the microbiota of some but not all individuals. It has been suggested that the nature of consumed isoflavones (glycosides versus aglycones) could influence the production of equol in humans 107 , thus highlighting the importance of the plant material source of isoflavones with respect to their biological activity. Equol has shown health-promoting effects in models of stroke-like injury, osteoporosis, cardiovascular disease, and menopause [108][109][110][111] and was recently found to alleviate toxicity elicited by the PD-related toxins MPP + and 6-OHDA in SH-SY5Y cells 112 114,115 , and equol was found to be highly permeable in an artificial BBB permeability assay 116 . Accordingly, we infer that the isoflavones and isoflavone-rich extracts examined in this study could potentially be of clinical benefit by reducing PD risk or slowing neurodegeneration in the brains of patients. The fact that daidzein and equol interfered with dopaminergic cell death at nanomolar concentrations in our primary midbrain culture model (Fig. 5C-E and G) implies that even a modest accumulation of these compounds in the brain could lead to neuroprotective effects in humans. Importantly, the ability of the extracts and individual isoflavones to mitigate neurotoxicity elicited by rotenone or A53T aSyn implies that these agents could potentially interfere with neurodegeneration in individuals with elevated PD risk triggered by a range of PD-related stresses. Consistent with this idea, we have obtained evidence from an ethnopharmacological study that a red clover extract is used as a form of traditional medicine by the Pikuni-Blackfeet Native Americans to treat PD-related symptoms 27 , and additional epidemiological data suggest that the consumption of isoflavones is associated with a reduced risk of neurodegenerative diseases 28, 29 .

A red clover extract activates the Nrf2/ARE antioxidant pathway and interferes with UPS function in cortical astrocytes.
A key result of this study was our finding that a red clover extract, but not a soy extract or the individual isoflavones daidzein and equol, activated Nrf2 signaling in primary cortical astrocytes. We infer that Nrf2 underwent nuclear translocation in cells treated with extract based on our EGFP imaging data (in the case of cells transduced with ARE-EGFP reporter virus) and qRT-PCR data, both of which showed evidence of the transcription of Nrf2-regulated genes. In addition, Nrf2 nuclear translocation was previously shown to occur in parallel with the activation of an AREluciferase reporter closely related to the ARE-EGFP reporter used here 117 .
There is conflicting evidence in the literature about the ability of isoflavones to activate the Nrf2/ARE pathway and induce the expression of Nrf2-regulated genes. For example, two isoflavone metabolites, tectorigenin and glycitein, were found to up-regulate HO1 and NAD(P)H Quinone Dehydrogenase 1 (NQO1) expression in a Nrf2-dependent manner in cortical astrocytes 118 .
Daidzein was shown to up-regulate HO1 in smooth muscle-derived cells but failed to increase quinone reductase (QR) activity in colon cancer cells 119 . In another study, daidzein and genistein were found to increase QR activity in different tissues of mice fed a daidzein-or genistein-enriched diet 120 . Interestingly, the latter study revealed variable tissue-and sex-dependent effects of isoflavone administration on glutathione-S-transferase activity 120 . These reported differences across various tissues suggest a possible cell type-specific response to isoflavones that could explain the lack of Nrf2 activation by the soy extract and individual isoflavones in cortical astrocytes ( Supplementary Fig. 4).
This is the first report of UPS inhibition by a red clover extract (Fig. 4A). A number of polyphenols, including isoflavones, have been shown by our group (Supplementary Fig. 5) and others to interfere with UPS activity 79,81,[121][122][123] . Genistein, a soy isoflavone, was reported to inhibit the chymotrypsin-like activity of the proteasome in a cancer cell model, likely by interacting with the proteasomal β5 subunit 123 . Our observation that a red clover extract interfered with GFPu degradation implies that polyphenols in this extract could induce activation of the Nrf2/ARE pathway by inhibiting UPS function. The concentration at which the extract induced a significant increase in the GFPu signal is higher than the concentration used for Nrf2 activation, implying that the sensitivity of the GFPu reporter assay is less than that of the ARE-EGFP assay, and, therefore, a low level of proteasome inhibition may be sufficient to induce Nrf2 signaling but not sufficient to cause a measurable amount of GFPu accumulation. In contrast, red clover polyphenols apparently do not activate Nrf2-mediated transcription via a mechanism involving redox cycling reactions leading to Keap1 oxidation, given that the antioxidant molecule NAC had no impact on the ability of the red clover extract to activate Nrf2 signaling in cortical astrocytes (Fig. 3) 48,60,124 . Finally, our observation that the red clover extract induced a modest up-regulation of Nrf2 mRNA levels ( Supplementary Fig. 3) suggests that this is an additional mechanism by which red clover polyphenols could activate Nrf2 signaling, consistent with similar effects reported for quercetin and resveratrol 88, 89 .

Activation of astrocytic Nrf2 signaling by a red clover extract depends on the brain region from which the astrocytes are prepared.
Another important outcome of our study was the finding that astrocytic Nrf2-mediated transcription was activated by a red clover extract in astrocytes prepared from rat cortex (Fig. 2C,D), but not in astrocytes prepared from rat midbrain or in mixed midbrain cultures (Supplementary Fig.   1). These observations are consistent with previous data showing that astrocytes from different brain regions differ in terms of their molecular profiles and their responses to different stimuli [125][126][127][128][129][130][131][132] .
For example, midbrain and cortical astrocytes were found to express different levels of glial glutamate transporter-1 (GLT-1) in response to corticosteroid stimulation 133 . The data presented here support previous findings relating to the heterogeneity of glial cells 125,130,134 by showing that astrocytes prepared from different brain regions respond differently to inducers of the Nrf2-mediated antioxidant response. Another explanation could be that red clover polyphenols undergo metabolic reactions that result in a loss of the extract's ability to modulate the Nrf2/ARE pathway in midbrain but not cortical astrocytes. Our data could also be explained by differences in the timing of Nrf2 activation among different types of astrocytes, although our observation that all of the astrocytic cultures examined here showed similar degrees of Nrf2-dependent signaling after treatment with curcumin for 24 h (Supplementary Fig. 2B) indicates that at least some forms of polyphenolmediated Nrf2 activation can occur with similar kinetics in astrocytes from different brain regions.
This observation also suggests that (i) curcumin and the red clover extract activate Nrf2 signaling via different mechanisms; or (ii) curcumin is resistant to metabolic reactions that interfere with Nrf2 activation by red clover polyphenols in mixed midbrain cultures. Finally, we note that the activation of our ARE-EGFP reporter derived from the mouse HO-1 gene presumably only reflects the expression of a subset of all 200+ Nrf2 targets. Accordingly, additional Nrf2-dependent transcriptional events could occur even in isoflavone-treated astrocytes that show no fluorescent signal in our reporter assay.
Collectively, our results suggest that the ability of isoflavone-rich extracts to activate astrocytic Nrf2 signaling varies in astrocytes and mixed cultures prepared from different brain regions. Our observation that the red clover and soy extracts exhibited opposite patterns of Nrf2 activation in rat cortical astrocytes versus human iPSC-derived astrocytes (Figs. 2 and 6 and Supplementary Fig. 4A) further highlights the point that isoflavone-rich extracts induce up-regulation of Nrf2-mediated transcription in a manner that varies across different types of astrocytic cultures.
Uncovering mechanisms underlying these brain region-and cell culture-specific responses has the potential to advance our understanding of the role of astrocytes in neuroprotection and enhance the impact of medicines designed to target the Nrf2/ARE pathway.
Lastly, because the red clover extract failed to activate Nrf2 signaling monitored using the ARE-EGFP reporter in primary midbrain cultures, we infer that the extract's protective effects against toxicity elicited by rotenone or A53T aSyn did not involve the up-regulation of astrocytic, Nrf2-mediated transcriptional events reflected by the reporter (e.g. HO-1 gene transcription) under our experimental conditions. Nevertheless, the extracts could potentially alleviate dopaminergic cell death in midbrain cultures exposed to PD-related insults via Nrf2 signaling mechanisms not reflected by our ARE-EGFP reporter, as discussed above.

Isoflavone-rich extracts rescue mitochondrial dysfunction.
O 2 consumption experiments revealed that the red clover and soy extracts alleviated rotenoneinduced deficits in mitochondrial electron transport in SH-SY5Y neuroblastoma cells (Fig. 7A).
These findings are consistent with previous data showing that soy isoflavones mitigated brain mitochondrial oxidative stress triggered by the amyloid-β peptide in rat models of AD 135,136 . Based on evidence that the neurotoxic effects of rotenone exposure and aSyn over-expression are related to mitochondrial functional deficits 9, 71, 94-96 , we infer that a rescue of these deficits by the red clover and soy extracts could play a key role in the extracts' ability to protect against these insults. Our finding that the red clover extract, but not the soy extract, preserved mitochondrial function at least in part by displacing rotenone from its binding site in complex I (Fig. 7B,C) suggests that red clover polyphenols can protect against rotenone neurotoxicity by interfering with the toxicant's ability to disrupt mitochondrial electron transport. Previous studies revealed that isoflavones such as genistein interact with the electron transport chain 137 , and genistein and daidzein were found to associate with the F 0 F 1 -ATPase/ATP synthase 138

An isoflavone-enriched soy extract protects against 6-OHDA toxicity in vivo.
Treatment of 6-OHDA-lesioned rats with Novasoy 400, a 40% (w/v) isoflavone standardized soy extract enriched in genistin, daidzein, and glycitin, led to an amelioration of 6-OHDA-induced motor dysfunction (Fig. 8). These findings suggest that soy isoflavones can mitigate nigrostriatal degeneration induced by 6-OHDA, potentially by rescuing mitochondrial dysfunction and activating antioxidant pathways in astrocytes, as seen in our cellular models of PD. Consistent with this idea, isoflavones such as genistein and equol have been shown to cross the BBB to reach target cell types 114,116 . The ability of a standardized soy extract to improve motor function in 6-OHDA-treated rats is in agreement with other published studies showing neuroprotective activities of individual soy isoflavones in animal models of PD. For example, the abundant soy isoflavone genistein was shown to increase the survival of neurons in the substantia nigra of rodents exposed to 6-OHDA 36 or MPTP 34 , and the daidzein metabolite equol protected Caenorhabditis elegans against toxicity elicited by MPP + 112 . Future studies will be focused on examining neuroprotective effects of Novasoy 400 via immunohistochemical analysis of brain sections from rats exposed to 6-OHDA and other PD-related insults.

Conclusion
In conclusion, isoflavone-rich red clover and soy extracts, and the individual isoflavones daidzein and equol, were found to alleviate neurotoxicity elicited by insults linked epidemiologically or genetically to PD. Equol showed potent neuroprotective activity at levels similar to those in the plasma of equol-producers, suggesting that there may be opportunities for the development of

Conflict of interest:
The authors declare no competing interest.
Graphical Abstract. The isoflavone-rich extracts red clover and soy and the individual isoflavones daidzein and equol protect neuronal cultures against environmental and genetic triggers of Parkinson's disease, and rescue motor deficits in rats exposed to the neurotoxin 6-OHDA.