Flavones 7,8-DHF, Quercetin, and Apigenin Against Tau Toxicity via Activation of TRKB Signaling in ΔK280 TauRD-DsRed SH-SY5Y Cells

Alzheimer’s disease (AD) is a progressive neurodegenerative disease with memory loss and cognitive decline. Neurofibrillary tangles (NFTs) formed by hyperphosphorylated Tau protein are one of the pathological hallmarks of several neurodegenerative diseases including AD. Heat shock protein family B (small) member 1 (HSPB1) is a molecular chaperone that promotes the correct folding of other proteins in response to environmental stress. Nuclear factor erythroid 2-like 2 (NRF2), a redox-regulated transcription factor, is the master regulator of the cellular response to excess reactive oxygen species. Tropomyosin-related kinase B (TRKB) is a membrane-bound receptor that, upon binding brain-derived neurotrophic factor (BDNF), phosphorylates itself to initiate downstream signaling for neuronal survival and axonal growth. In this study, four natural flavones such as 7,8-dihydroxyflavone (7,8-DHF), wogonin, quercetin, and apigenin were evaluated for Tau aggregation inhibitory activity and neuroprotection in SH-SY5Y neuroblastoma. Among the tested flavones, 7,8-DHF, quercetin, and apigenin reduced Tau aggregation, oxidative stress, and caspase-1 activity as well as improved neurite outgrowth in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. Treatments with 7,8-DHF, quercetin, and apigenin rescued the reduced HSPB1 and NRF2 and activated TRKB-mediated extracellular signal-regulated kinase (ERK) signaling to upregulate cAMP-response element binding protein (CREB) and its downstream antiapoptotic BCL2 apoptosis regulator (BCL2). Knockdown of TRKB attenuated the neuroprotective effects of these three flavones. Our results suggest 7,8-DHF, quercetin, and apigenin targeting HSPB1, NRF2, and TRKB to reduce Tau aggregation and protect cells against Tau neurotoxicity and may provide new treatment strategies for AD.


INTRODUCTION
Neurodegenerative disorders tauopathies, including the most common Alzheimer's disease (AD), are characterized by abnormal hyperphosphorylation of microtubule-associated protein Tau that leads to the formation of neurofibrillary tangles (NFTs) and causes gain of toxic function (Iqbal et al., 2005). Tau is mainly expressed in neuronal axons, where it promotes assembly and bundling of microtubules, thereby regulating vesicular transport and apoptosis (Lee et al., 2001). Phosphorylation of Tau has been proposed as the link between oxidative stress, mitochondrial dysfunction, and synaptic failure during early stages of AD (Mondragón-Rodríguez et al., 2013). In retinal ganglion cells of human P301S Tau transgenic mice with early tauopathy, the impairment of tropomyosin-related kinase B (TRKB) signaling is triggered by Tau pathology and mediates the Tau-induced dysfunction of visual response (Mazzaro et al., 2016). In addition, the accumulated asparagine endopeptidasecleaved N368 Tau N-terminal fragment binds the TRKB receptor on its C terminus and antagonizes neurotrophic signaling to trigger neuronal apoptosis (Xiang et al., 2019).
Tropomyosin-related kinase B, a member of the neurotrophic tyrosine receptor kinase family, is a high-affinity receptor for brain-derived neurotrophic factor (BDNF) (Soppet et al., 1991). Upon BDNF binding, TRKB phosphorylates itself and members of the mitogen-activated protein kinase (MAPK) pathway initiate intracellular signaling cascades (Levine et al., 1996). TRKB is highly expressed in adult hippocampus (Nakagawara et al., 1995), an area of the brain involved in learning and memory. Rapid activation of extracellular signal-regulated kinase (ERK), a member of the MAPK family, by BDNF/TRKB signaling induces phosphorylation of cAMP-response element binding protein (CREB) (Bonni et al., 1999) to stimulate expression of antiapoptotic BCL2 apoptosis regulator (BCL2) (Riccio et al., 1999) for neuronal survival. As a significant risk factor for AD (Coppola et al., 2012), p.A152T Tau variant alters Tau function and toxicity via impairing retrograde axonal transport of synaptic vesicles (Butler et al., 2019). As retrograde axonal transport of endosomes mediated by TRKB signaling is essential for dendrite growth of cortical neurons (Zhou et al., 2012), BDNF/TRKB potentiation would be protective in AD.
Structurally, Tau is a prototypical natively unfolded protein (Schweers et al., 1994). Tau binds microtubules through C-terminal highly conserved 18-amino acid repeat domains (Tau RD ), which reside at the core of paired helical filaments of NFT (Goedert et al., 1989). The Tau RD with the deletion mutation K280 is highly prone to spontaneous aggregation (Khlistunova et al., 2006). Heat shock protein family B (small) member 1 (HSPB1) can prevent pathological misfolding of Tau by altering the conformation of hyperphosphorylated Tau and rescue hyperphosphorylated Tau-mediated cell death (Shimura et al., 2004). Accumulation of misfolded proteins can cause oxidative stress and compromise nuclear factor erythroid 2-like 2 (NRF2) to incur early events in the pathogenesis of AD (Mota et al., 2015). NRF2 activators have therapeutic effects in AD animal models and in cultured human cells that express the pathology of AD (Bahn and Jo, 2019).

Test Compounds and Biochemical
Analysis of Tau Aggregation Inhibition 7,8-dihydroxyflavone, wogonin, quercetin, and apigenin were purchased from Sigma-Aldrich, St. Louis, Mosby, MO, United States. Congo red (Sigma-Aldrich, St. Louis, Mosby, MO, United States), known to bind to the cross β-sheet structure of amyloid fibrils (Sipe and Cohen, 2000), was included for comparison. For biochemical Tau aggregation inhibition test, bacterial expressed proaggregator K280 Tau RD (Gln 244 -Glu 372 of 441-residue human Tau) was prepared as described . K280 Tau RD protein (20 µM in final 50 µl) was incubated with tested compounds (5-10 µM in 150 mM sodium chloride (NaCl) and 20 mM tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl), pH 8.0) at 37 • C for 48 h. Then, thioflavin T (5 µM final concentration; Sigma-Aldrich, St. Louis, Mosby, MO, United States), a dye exhibiting enhanced fluorescence upon binding to diverse types of amyloid fibrils (Biancalana and Koide, 2010), was added and incubated for 25 min at room temperature. The formed aggregates reflected by thioflavin T fluorescence intensity was recorded at excitation 420 nm and emission 485 nm by using the FLx800 Fluorescence Microplate Reader (BioTek Instruments Incorporation, Winooski, VT, United States). Half maximal effective concentration (EC 50 ) was calculated using the interpolation method.
For reactive oxygen species (ROS) measurement, dichlorodihydro-fluorescein diacetate (DCFH-DA) (10 µM; Invitrogen, Carlsbad, CA, United States), a fluorogenic dye that measures hydroxyl, peroxyl, and other ROS activity within cells (Aranda et al., 2013), was added to the cells on day 8 and incubated at 37 • C for 30 min. ROS in cells was measured using the High-Content Imaging System, with excitation/emission wavelengths at 482/536 nm.

Real-Time PCR Analysis
As described, K280 Tau RD -DsRed SH-SY5Y cells were seeded on a 6-well plate (5 × 10 5 /well), with retinoic acid addition on day 1, treated with tested compounds (5 or 10 µM), and induced K280 Tau RD -DsRed expression with doxycycline on day 2. On day 8, cells were collected and total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, United States). The RNA was reverse-transcribed using the SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, United States). Real-time quantitative PCR was performed using 50 ng complementary DNA (cDNA) with the customized Assaysby-Design probe for DsRed (Chang et al., 2017) and the TaqMan fluorogenic probe for hypoxanthine phosphoribosyltransferase 1 (HPRT1) (4326321E) using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). Fold change was calculated using the formula 2 Ct , C T = C T (HPRT1) − C T (DsRed), in which C T indicates cycle threshold.

Neurite Outgrowth Analysis
As described, K280 Tau RD -DsRed SH-SY5Y cells were seeded on a 24-well plate (5 × 10 4 cells/well), with induced neuronal differentiation on day 1, treated with test compounds (5 or 10 µM), and induced K280 Tau RD -DsRed expression on day 2. After 7 days, the cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 10 min. After being permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 3% bovine serum albumin in PBS for 20 min, cells were stained with tubulin beta 3 class III (TUBB3) primary antibody (1:1,000; Covance, Princeton, NJ, United States) at 4 • C overnight, followed by goat antirabbit Alexa Fluor R 555 secondary antibody (1:1,000; Invitrogen, Carlsbad, CA, United States) at room temperature for 3 h. After nuclei were stained with 4 ,6-diamidino-2-phenylindole (DAPI) (0.1 µg/ml; Sigma-Aldrich, St. Louis, Mosby, MO, United States) for 30 min, neuronal images from at least 60 individual fields (150-250 neurons per field) per experiment were captured at excitation/emission wavelengths of 531/593 nm using the ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices, Sunnyvale, CA, United States). Neurite total length (µm), process (the number of primary neurites defined as the segments originating from the cell body of a neuron), and branch (the number of secondary neurites extending from primary neurites) were analyzed using the MetaXpress Neurite Outgrowth Application Module (Molecular Devices, Sunnyvale, CA, United States). For each sample, around 6,000 cells were analyzed in each of 3 independent experiments.
Caspase-1, Lactate Dehydrogenase, and Acetylcholinesterase Assays K280 Tau RD -DsRed SH-SY5Y cells were seeded in a 6well plate (5 × 10 5 /well) and treated with retinoic acid, test compound, and doxycycline as described. On day 8, cells were collected and lysates were prepared by 6 freeze/thaw cycles. After centrifugation to collect supernatant, caspase-1 activity in 50 µg cell extracts was measured using the ICE Fluorometric Assay Kit (BioVision, Milpitas, CA, United States). The mixture was incubated for 2 h at 37 • C and caspase-1 activity was measured with excitation/emission wavelengths at 400/505 nm (FLx800 Fluorescence Microplate Reader; BioTek Instruments Incorporation, Winooski, VT, United States). In addition, the collected cells were lysed by sonication. Acetylcholinesterase (AChE) activity in supernatant was determined using AChE Activity Assay Kit (Sigma-Aldrich, St. Louis, Mosby, MO, United States) with 10 µg cell extracts. The mixture was incubated for 2-10 min at room temperature and absorbance at 412 nm was measured using the Multiskan GO Microplate Spectrophotometer Reader (Thermo Fisher Scientific, Waltham, MA, United States). The release of lactate dehydrogenase (LDH) in culture medium was examined by using the LDH Cytotoxicity Assay Kit (Cayman, Ann Arbor, MI, United States). The absorbance was read at 490 nm with the Multiskan GO Microplate Spectrophotometer Reader.

Ribonucleic Acid Interference
For TRKB knockdown in K280 Tau RD -DsRed SH-SY5Y cells, lentivirus carrying TRKB-targeting (TRCN0000002243, TRCN0000002245, and TRCN0000002246) and negative control scrambled (TRC2.Void) short hairpin RNA (shRNA) were obtained from the National RNAi Core Facility, institute of molecular biology/genomic research center, Academia Sinica, Taipei, Taiwan. On day 1, cells were plated on 24-well plates in the presence of retinoic acid as described. On day 2, the cells were infected with lentivirus (3 multiplicity of infection for each shRNA) in medium with polybrene (8 µg/ml; Sigma-Aldrich, St. Louis, Mosby, MO, United States). On day 3, the culture medium was changed and the cells were pretreated with Congo red (10 µM), 7,8-DHF (5 µM), quercetin (5 µM), or apigenin (10 µM) for 8 h, followed by induction of K280 Tau RD -DsRed expression. On day 9, the cells were collected for TRKB protein analysis or analyzed for neurite outgrowth as described.

Statistical Analysis
Data are presented as mean ± SD. 3 independent tests in 2 or 3 biological replicates were performed in each experiment. Differences between groups were evaluated using the two-tailed Student's t-test or the one-way ANOVA with the post hoc Tukey's test where appropriate. p < 0.05 indicate a statistically significant difference. Tau RD aggregation inhibition of tested compounds (5-10 µM) by the thioflavin T assay (n = 3). p-values: comparisons between 0-h and 48-h incubation ( ### p < 0.001) or between with and without compound addition (*p < 0.05, **p < 0.01, and ***p < 0.001) (one-way ANOVA with the post hoc Tukey's test). Shown below are the EC 50 values.

Tested Flavones, Radical Scavenging, and Biochemical Tau Aggregation Inhibition
Natural flavones such as 7,8-DHF, wogonin, quercetin, and apigenin ( Figure 1A) were examined. Oxidative stress has been recognized as a contributing factor to the progression of AD and preventing oxidative stress is considered as a treatment approach for AD (Poprac et al., 2017). Therefore, the radical scavenging activity of the three flavones (10-80 µM) was first examined. While wogonin and apigenin displayed no radical scavenging activity, 7,8-DHF and quercetin had EC 50 values of 24 and 25 µM, respectively ( Figure 1B).

Tropomyosin-Related Kinase B Binding Prediction
The strengths and conformation of wogonin, quercetin, and apigenin binding with neurotrophin-binding domain d5 of TRKB were calculated using 7,8-DHF as a control. As shown in Figure 2, the computations predicted docking scores of 44.67, 44.08, 49.77, and 47.91 for 7,8-DHF, wogonin, quercetin, and apigenin, respectively. Quercetin had the top interacting docking score with TRKB receptor among the 3 flavones examined.
high docking scores of the tested compounds binding to the neurotrophin-binding domain d5 of TRKB predicted by the computations (Figure 2). The observed neuroprotective effect under the knockdown of TRKB by these flavones may be also attributed to the increased NRF2 to promote neurite outgrowth (Yang et al., 2015). K280 Tau RD -DsRed. As described, cells were seeded with RA, treated with Congo red (10 µM), 7,8-DHF (5 µM), quercetin (5 µM), or apigenin (10 µM), and induced K280 Tau RD -DsRed expression with doxycycline for 6 days. On day 8, (A) caspase-1 activity, (B) lactate dehydrogenase (LDH) release, (C) AChE activity, and (D) neurite length, process, and branch were analyzed (n = 3). To normalize, the relative caspase-1, LDH release, or AChE activity of uninduced cells (Dox -) was set as 100%. Shown on the bottom half of panel (D), there were images of TUBB3 (green)-stained cells, with nuclei counterstained with DAPI (blue) and segmented images with multicolored mask to assign each outgrowth to a cell body for neurite outgrowth quantification. Process and branches in uninduced cells were marked with red and white arrows, respectively. p-values: comparisons between induced vs. uninduced cells ( # p < 0.05) or compound-treated vs. untreated cells (*p < 0.05, **p < 0.01, ***p < 0.001) (one-way ANOVA with the post hoc Tukey's test).
Various natural flavones display antioxidant activity (Pietta, 2000). The majority of neurodegenerative diseases are speculated to originate from cumulative oxidative stress caused by deposition of abnormal aggregated proteins (Grimm et al., 2011). There is clear evidence that ROS is highly upregulated in the brain of tauopathies of the patients and ROS also directly promotes Tau modifications (Haque et al., 2019). Compounds with antioxidative potential may directly serve as chemical chaperone to suppress protein aggregates or quench free oxygen radicals. All the three flavones displayed some degree of chemical chaperone activity to enhance the folding and/or stability of proaggregator Tau (Figure 1C). Structural requirements of flavonoids for appreciable radical scavenging activity have been established (Seyoum et al., 2006). Without o-dihydroxy group (catechol structure) in the benzene ring, apigenin has poor antioxidant capacity for scavenging free radicals as assessed using stable radical DPPH ( Figure 1B).
An alternative way to decrease cellular ROS is by enhancing antioxidative signaling such as NRF2 pathway. Stabilized following oxidative stress, NRF2 induces the expression of antioxidants as well as cytoprotective genes (Vomund et al., 2017). Reduced nuclear levels of NRF2 are observed in postmortem brains of patients with AD (Ramsey et al., 2007) and NRF2 inducer carnosic acid improves learning and memory in 3 × Tg-AD mice (Lipton et al., 2016). In this study, all the three flavones enhanced the expression of NRF2 (Figure 3E), supporting the role of 7,8-DHF, quercetin, and apigenin in activating the adaptive response to reduce oxidative stress. The NRF2 expression can be upregulated by AKT serine/threonine kinase 1 (AKT), which is one of the downstream proteins of TRKB activation (Yoo et al., 2017). Therefore, the NRF2 in the K280 Tau RD cell model treated with the three flavones may be increased through TRKB activation, at least partially.
Heat-shock proteins such as HSP90, HSP40, and HSPB1 had been speculated to function as regulators of soluble Tau protein levels (Sahara et al., 2007). As all the three flavones enhanced the expression of HSPB1 for chaperoning misfolded Tau (Figure 3E), 7,8-DHF, quercetin, and apigenin may also reduce the Tau-associated ROS through eliminating the misfolded Tau deposits. HSPB1 is transcriptionally upregulated by heat shock transcription factor 1 that can be enhanced by NRF2 (Paul et al., 2018). The HSPB1 expression in the K280 Tau RD cell model treated with the tested compounds may be activated by increased NRF2. However, further studies are required to consolidate the assumption.
Acetylcholinesterase, an enzyme breaking down the neurotransmitter acetylcholine, is a feasible therapeutic target for treatment of AD (Akıncıoglu and Gülçin, 2020). Currently AChE inhibitors such as donepezil, rivastigmine, and galantamine are commonly used to increase the level and duration of acetylcholine and facilitate cholinergic transmission in AD (Marucci et al., 2021). It has been shown that AChE activity as well as choline acetyltransferase is reduced in the cerebral cortex of patients with AD and tauopathy, indicating degenerated cholinergic neurons in both the diseases (Shinotoh et al., 2000;Hirano et al., 2006Hirano et al., , 2018. However, the AChE activity is increased in frontotemporal dementia with parkinsonism-17 human tau transgenic mice (Silveyra et al., 2012). The findings of AChE activity in human tauopathy and the mouse model are not consistent. Our cell model that did not show significant changes in AChE activity may indicate that AChE activity is still preserved or partially impaired in this model and that inhibiting AChE activity may rescue acetylcholine levels. Among the three flavones examined, only apigenin reduced AChE activity in SH-SY5Y cells expressing proaggregator Tau ( Figure 4C). Studies have shown that oxidative stress may induce AChE activity . However, given that all the three flavones in this study can reduce ROS, the reduced AChE activity only by apigenin may be attributed to some unknown mechanism. In the past years, intensive study efforts have been made for developing multitarget anti-Alzheimer compounds that hit several key pathogenic factors of the disease. Hybrid compounds combining a unit of potent and selective AChE inhibitor huprine Y with the 4-hydroxy-3-methoxyphenylpentanone moiety of natural antioxidant 6-shogaol have been documented (Pérez-Areales et al., 2014). In addition, herbal formulations with anticholinesterase and antioxidant activity could be benefit to memory enhancement (Nwidu et al., 2018). Being a dual antioxidant and anticholinesterase agent with tau antiaggregating property (Figures 1, 3, 4), apigenin emerges as an interesting multitarget anti-AD agent.
Upon BDNF binding, TRKB dimerized and phosphorylated to initiate intracellular signaling such as ERK, leading to activation of transcription factor CREB and downstream antiapoptotic BCL2 for neuronal survival (Walton and Dragunow, 2000). The level of phosphorylated CREB is decreased in the hippocampus of old rats with spatial memory deficits (Kudo et al., 2005;Williams et al., 2008;Xu et al., 2010). BCL2 binds to and inactivates BAX, thereby inhibiting apoptosis (Jonas, 2009). In 3 × Tg-AD mice, Tau provokes downregulation of BCL2 and increases level of BAX to lead to degeneration of cochlear spiral ganglion neurons (Wang and Wu, 2021). In our SH-SY5Y cell model, induction of proaggregated K280 Tau RD expression downregulated BCL2 and upregulated BAX, whereas 7,8-DHF, quercetin, and apigenin rescued changes in these gene expression (Figure 4). Although Congo red was used as a control, it did not rescue changes of p-CREB, BCL2, and BAX, which is probably due to its modest neuroprotection effect. In contrast, under the same condition, the tested flavones showed significant improvements in p-CREB, BCL2, and BAX, suggesting their promising therapeutic potential. As crosstalk between pathological Tau phosphorylation and mitochondrial dysfunction exists (Guha et al., 2020) and BCL2 counteracts the mitochondria dysfunction-mediated apoptosis, investigation of mitochondria-related apoptotic pathways through caspase-9 and caspase-3 activation may shed light on the mechanism of how quercetin and apigenin provide antiapoptotic effect on K280 Tau RD cells. There are still other TRKB downstream pathways such as phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR. In the future, investigating if PI3K/AKT/mTOR is also involved in the neuroprotective mechanism of these flavones is warranted. Besides, although our previous study has shown the neuroprotection effects of 7,8-DHF in the primary hippocampal primary neurons and mouse models induced by Aβ (Fan et al., 2020), whether the other two flavones apigenin and quercetin also provide the similar effects either in primary neurons and mouse models should be examined in the future.
In conclusion, our results show that, in addition to 7,8-DHF, quercetin and apigenin activate TRKB signaling to upregulate downstream CREB and BCL2 expressions. In the proaggregator Tau AD cell model, these flavones improve neurite outgrowth, reduce caspase-1 and/or AChE activities by activating TRKB, as well as ameliorating ROS. As multiple pathogenic pathways are involved in AD, the potential of these flavones targeting multiple pathways may have a significant perspective for developing anti-AD drug. The effect of quercetin and apigenin as TRKB agonists should be validated in AD animal models. Assays of mitochondrial function, especially in the light of increased BCL2 expression, would provide a better connection between the reported signaling mechanisms and resulting changes in cell physiology. Also, binding of quercetin and apigenin to the TRKB receptor should be measured using surface plasmon resonance to consolidate the action as agonists of TRKB.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
N-NC, T-HL, and Y-ST contributed to the execution of experiments, data analysis, and interpretation. Y-CS, K-HC, C-YL, HH-L, and M-TS contributed to review and editing. C-MC and G-JL-C contributed to the concept and design, data analysis and interpretation, obtained funding, and wrote and finalized the manuscript. All authors have read and agreed to the published version of the draft of the manuscript.

FUNDING
This study was supported by the grants 108-2320-B-003-001 and 108-2320-B-182A-001 from the Ministry of Science and Technology, Taiwan.