New Synthetic 3-Benzoyl-5-Hydroxy-2H-Chromen-2-One (LM-031) Inhibits Polyglutamine Aggregation and Promotes Neurite Outgrowth through Enhancement of CREB, NRF2, and Reduction of AMPKα in SCA17 Cell Models

Spinocerebellar ataxia type 17 (SCA17) is caused by a CAG/CAA expansion mutation encoding an expanded polyglutamine (polyQ) tract in TATA-box binding protein (TBP), a general transcription initiation factor. Suppression of cAMP-responsive element binding protein- (CREB-) dependent transcription, impaired nuclear factor erythroid 2-related factor 2 (NRF2) signaling, and interaction of AMP-activated protein kinase (AMPK) with increased oxidative stress have been implicated to be involved in pathogenic mechanisms of polyQ-mediated diseases. In this study, we demonstrated decreased pCREB and NRF2 and activated AMPK contributing to neurotoxicity in SCA17 SH-SY5Y cells. We also showed that licochalcone A and the related in-house derivative compound 3-benzoyl-5-hydroxy-2H-chromen-2-one (LM-031) exhibited antiaggregation, antioxidative, antiapoptosis, and neuroprotective effects in TBP/Q79-GFP-expressing cell models. LM-031 and licochalcone A exerted neuroprotective effects by upregulating pCREB and its downstream genes, BCL2 and GADD45B, and enhancing NRF2. Furthermore, LM-031, but not licochalcone A, reduced activated AMPKα. Knockdown of CREB and NRF2 and treatment of AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside), an AMPK activator, attenuated the aggregation-inhibiting and neurite outgrowth promoting effects of LM-031 on TBP/Q79 SH-SY5Y cells. The study results suggest the LM-031 as potential therapeutics for SCA17 and probable other polyQ diseases.


Introduction
Hereditary spinocerebellar ataxia types (SCAs) 1, 2, 3, 6, 7, and 17 are among a group of inherited neurodegenerative diseases caused by the expansion of unstable trinucleotide (CAG) repeats encoding expanded polyglutamine (polyQ) tracts [1]. PolyQ-mediated diseases also include Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), and dentatorubral-pallidoluysian atrophy (DRPLA). SCA17 is an autosomal dominant ataxia caused by an allele con-taining expanded repeats longer than 43 in the TATAbox binding protein (TBP) gene, a transcription initiation factor [2]. Protein containing an expanded polyQ tract tends to change conformation and form intranuclear and cytoplasmic aggregates, thereby causing several pathologic processes including transcriptional dysregulation, mitochondrial abnormalities, oxidative stress, axonal transport defect, chaperone-proteasome impairment, autophagolysosome dysfunction, and unfolded protein response (UPR) in the endoplasmic reticulum (ER) [1,3]. Transcriptional dysregulation is one of the main pathogenic mechanisms of polyQ diseases [4]. Several lines of evidence have shown that nuclear localization of polyQ containing protein interferes with nuclear transcription factors and cofactors leading to cellular toxicity. CBP (cAMP-responsive element binding protein-(CREB-) binding protein), a cofactor for CREB-dependent transcriptional activation, has been shown to colocalize with the mutant polyQ containing protein in cell and mouse models and human HD brains [5]. Suppression of CREB-dependent transcription has been shown in a HD cell model, HD and SCA3 mice, and human HD brains [6][7][8][9][10]. Therefore, enhancement of CREB-mediated transcriptional activation is considered as a potential therapeutic strategy for polyQ diseases [11][12][13][14][15][16]. Since transcriptional dysregulation has been shown to play a role in pathogenic mechanisms of SCA17 [17], we proposed that CREB-mediated gene expression is downregulated in SCA17 and compounds augmenting CREB activation can rescue the cytotoxicity.
Increased oxidative stress induced by mutant polyQ protein, which results in cell death in vitro [18,19] and in vivo [20,21], has been shown. Antioxidants can ameliorate the aggregation and cytotoxicity in models of polyQ diseases [22][23][24][25][26]. The major pathway responsive to oxidative stress to protect cells is the nuclear factor erythroid 2-related factor 2 (NRF2) and the antioxidant response elements (AREs) signaling [27]. The target genes regulated by NRF2 are belonging to the endogenous phase II antioxidative enzymes. NRF2 activation can mitigate a number of neurodegenerative diseases including HD [28]. We and other researchers have shown that NRF2 expression is impaired in SCA3 and SCA17 models, and agents enhancing NRF2 rescue the phenotypes induced by mutant polyQ [2,22,[29][30][31][32]. Taken together, we planned to examine more compounds that may activate NRF2 in our SCA17 cell models.
AMP-activated protein kinase (AMPK) is a serine/threonine kinase that plays a mandatory role in maintaining cellular metabolic homeostasis. AMPK is regulated by the cellular adenylate charge and is activated in response to energy deficiency in cells [33]. AMPK consists of three subunits (α, β, and γ) and is mainly activated through the phosphorylation of the threonine residue 172 (Thr172) of α subunit [34]. The activity of AMPK is regulated by several kinases including calmodulin-dependent protein kinase kinase (CaMKK), liver kinase B1 (LKB1), TGF-β-activated kinase 1 (TAK1), cAMP-dependent kinase (PKA), and Ca 2+ /calmodulindependent protein kinase II (CaMKII) [35]. The function of AMPK is multifold. The interactions between oxidative stress and AMPK are complex and may play a different role under different conditions. AMPK is activated to protect a rat dental pulp cell line under oxidative stress [36]. AMPK activation reverses decreased cell viability induced by ageing-related oxidative stress [37] and also protects cells from oxidative stress-induced senescence via autophagic flux restoration [38]. A previous study also has shown that mitochondrial reactive oxygen species (ROS) physiologically activate AMPK, which induce an antioxidant program that regulates mitochondrial homeostasis and cellular metabolic balance [39]. However, Ju and colleagues have shown that mutant huntingtin (mHTT) abnormally activates AMPK-α1 via a CaMKII-dependent pathway and exerts a detrimental effect on neuronal survival [40]. The authors further showed that increased ROS and the activated AMPK-α1 act in a vicious cycle contributing to the mHTT-induced neuronal death in the striatum of HD mice [41]. Since previously our SCA17 models displayed increased oxidative stress [29,32,42], we planned to investigate the role of AMPK in the pathogenesis of SCA17 and if aggregation-inhibitory compounds act on the AMPK pathway.
We have also previously shown that licochalcone A and five related in-house derivative LM compounds exhibited antiaggregation, antioxidant, and neuroprotective effects against Aβ toxicity by enhancing the NRF2-related antioxidant and CREB-dependent survival pathway [43]. Therefore, we tested the effects of licochalcone A and these LM compounds targeting these pathways in TBP/Q 79 -GFP-expressing cell models.
2.2. Compound Cytotoxicity. Compound cytotoxicity was assessed by colorimetric assay measuring cell metabolic activity. Briefly, TBP/Q 79 -GFP 293 and SH-SY5Y cells were seeded on a 48-well plate (5 × 10 4 /well), grown for 20 h, and treated with licochalcone A or LM compounds (0.1-100 μM). After one day, cell viability was measured based on reduction of 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and the absorbance of the insoluble purple formazan product at OD 570 nm was read by a FLx800 fluorescence microplate reader (Bio-Tek, Winooski, VT, USA). Half-maximal inhibitory concentration (IC 50 ) was defined as the concentration of compounds required for the reduction of 570 nm signals by 50%. induction of TBP/Q 79 -GFP expression followed. On day 9, the cells were collected for NRF2 and CREB protein analysis or stained with Hoechst 33342 and analyzed for aggregation and neurite outgrowth as described.
On day 2, the cells were pretreated with LM-031 or licochalcone A (100 nM) for 4 h; AICAR (0.1 mM) treatment and TBP/Q 79 -GFP induction followed. On day 8, the cells were collected for AMPKα and pAMPKα protein analysis or stained with Hoechst 33342 and analyzed for aggregation and neurite outgrowth as described.
For filter trap assay to quantify polyQ aggregates, the purified TBP protein was incubated with tested compound (100 μM) at 37°C for 24 h as described previously. In brief, protein (1 μg) was diluted in 2% SDS in PBS and filtered through a cellulose acetate membrane (0.2 μm pore size; Merck, Kenilworth, NJ, USA) preequilibrated in 2% SDS in PBS on a dot-blot filtration unit (Bio-Rad Laboratories, Hercules, CA, USA). After three washes with 2% SDS buffer, the membrane was blocked in PBS containing 5% nonfat dried milk and stained with anti-TBP antibody (1 : 1000; Santa Cruz Biotechnology). The immune complexes containing aggregates on the filter were detected as described.
2.11. Statistical Analysis. For each data set, the experiments are performed three times and data were expressed as the means ± standard deviation (SD). Differences between groups were evaluated using Student's t test (comparing two groups) or one-way analysis of variance with a post hoc LSD test where appropriate (comparing several groups). p values lower than 0.05 were considered statistically significant. 50 Cytotoxicity. Licochalcone A and five related in-house LM compounds were tested (Figure 1(a)). The MTT assay was performed using uninduced TBP/Q 79 -GFP 293 and SH-SY5Y cells following treatment with the test compounds (0.  (Figure 2(a)). While no TBP/Q 79 -GFP expression was seen in the absence of doxycycline (data not shown), TBP/Q 79 -GFP aggregates were readily seen in the induced cells (Figure 2(b)). Representative microscopy images of TBP/Q 79 -GFP aggregation in untreated cells or after licochalcone A or LM-031 treatment (100 nM) were shown in Figure 2(c). As a positive control, SAHA at 100 nM significantly reduced the TBP/Q 79 -GFP aggregation to 82% (p < 0:001) compared with untreated cells (100%) (Figure 2 Abnormal TBP-containing polyQ expansion has been shown to increase cellular ROS level [42]. To evaluate whether licochalcone A or LM compounds reduced oxidative stress in TBP/Q 79 -GFP 293 cells, the cellular ROS production was measured. As shown in Figure 2(e), significantly increased ROS production (179% of control, p = 0:001) was observed in cells with induced TBP/Q 79 -GFP expression (+Dox) for 6 days (33.8-fold expression, p < 0:001). With the similar induced green fluorescence (34.1-34.9-fold, p > 0:05), the test licochalcone A and LM compounds (100 nM) significantly ameliorated oxidative stress induced by TBP/Q 79 -GFP expression (ROS fluorescence: from 349 to 277-247, p < 0:001). 3.4. Molecular Targets of LM-031 and Licochalcone A in SCA17 SH-SY5Y Cell Model. CREB is a transcription factor binding to cAMP-responsive elements to promote transcription of BCL2 (BCL2 apoptosis regulator) [51] and GADD45B (growth arrest and DNA damage-inducible beta) [52,53] genes for neuronal survival. Phosphorylation at Ser-133 induces CREB-mediated transcription of BCL2 and GADD45B. NRF2 is a master regulator of the antioxidant response through regulating the expression of phase II antioxidant and detoxification genes [27]. Both CREB and NRF2 have been shown to suppress polyQ-mediated toxicity in Drosophila [12,31]. As the neuroprotective effects of LM-031 and licochalcone A in Aβ-GFP-expressing SH-SY5Y cells had been linked to CREB and NRF2 pathways [43], we examined the expressions of CREB, pCREB (S133), BCL2, GADD45B, and NRF2 by immunoblotting using specific antibodies.     7 Oxidative Medicine and Cellular Longevity differentiated SH-SY5Y cells significantly attenuated phosphor/total ratio of CREB (85% of control, p = 0:044) and the downstream BCL2 and GADD45B (66-70% of control, p = 0:003 -0:002). This attenuation was rescued by the treatment with LM-031 and licochalcone A: increased to 110% for pCREB/CREB (p = 0:005), 125-118% for BCL2 (p < 0:001), and 104-105% for GADD45B (p = 0:001) ( Figure 4). In response to the antiapoptotic BCL2 change, the addition of LM-031 and licochalcone A significantly reduced the expression of proapoptotic BAX (BCL2 associated X, apoptosis regulator) (from 156% to 103%, p < 0:001). In addition, induction of TBP/Q 79 -GFP significantly reduced NRF2 expression (72% of control, p < 0:001). This reduction was also rescued by the treatment with LM-031 and licochalcone A (increased to 96-100%, p < 0:001) (Figure 4).

Neuroprotective Effects of LM-031 and Licochalcone
AMPK is a major energy sensor that maintains cellular energy homeostasis in the brain [54]. In Huntington's disease, mutant huntingtin protein induces oxidative stress to lead to aberrant activation of AMPKα1 and neuronal atrophy [40,41]. As increased ROS was associated with TBP/Q 79 -GFP expression (Figure 2(e)), we examined the effects of LM-031 and licochalcone A on the alternation of AMPK. As also shown in Figure 4, phosphor/total ratio of AMPKα was significantly increased after induction of TBP/Q 79 -GFP expression (170% of control, p = 0:003), whereas treatment with LM-031, but not licochalcone A, reduced the phosphor/total ratio of AMPKα (from 170% to 130%, p = 0:034).

Discussion
Up to now, no effective treatments are available for polyQinduced diseases including SCA17. The revelation of pathogenic mechanisms is the key to the development of therapeutics. In this study, we have shown impaired CREB-dependent transcription and NRF2-ARE pathway and increased AMPK activation in TBP/Q 79 -GFP-expressing SH-SY5Y cells. Our results also demonstrate the neuroprotective effects of licochalcone A and LM-031 on SCA17 SH-SY5Y cell model.
The licochalcone A and five LM compounds displayed a low cytotoxicity in uninduced TBP/Q 79 -GFP 293 and SH-SY5Y cells with IC 50 ≥ 45 μM as shown in Figure 1. Compound with low cytotoxicity is crucial for its future clinical application to treat diseases. The licochalcone A/LM compounds also demonstrated significant aggregationinhibitory as well as ROS-reducing effects in TBP/Q 79 -GFPexpressing 293 cells. Among tested LM compounds, LM-031 showed a better aggregation-inhibitory effect than SAHA, indicating its great potential as a therapeutic compound. Such aggregation-inhibitory effect is also found in TBP/Q 79 -GFP SH-SY5Y cells. In addition, LM-031 and licochalcone A displayed neuroprotection via not only rescuing neurite outgrowth deficits but also reducing caspase 3 activity. Further mechanism studies showed that downregulated NRF2, pCREB/CREB, and its downstream genes BCL2 and GADD45B in TBP/Q 79 -GFP SH-SY5Y cells could be upregulated by LM-031/licochalcone A. In addition, increased phosphor/total ratio of AMPKα induced by TBP/Q 79 -GFP expression was diminished by LM-031.
In order to know whether LM-031/licochalcone A exerted neuroprotection through acting on CREB, NRF2, and AMPK pathways, knockdown of NRF2 and CREB and AICAR was applied to the SCA17 SH-SY5Y cell model. The effects of reduced aggregation and promoted neurite outgrowth by LM-031/licochalcone A were attenuated by knockdown of NRF2 and CREB. These results indicate that LM-031/licochalcone A protects neurons from neurotoxicity induced by mutant TBP via targeting NRF2 and CREB pathway, both of which are compromised in SCA17.
Several lines of evidence have suggested that targeting NRF2 could be a potential therapeutic strategy for neurodegenerative diseases [56]. Indeed, agents activating NRF2, such as Gardenia jasminoides, Glycyrrhiza inflata, 3-alkyl luteolin, caffeic acid, and resveratrol are beneficial to different models of polyQ diseases including SCA3, HD, and SBMA [22,30,31,57,58]. Our study results provide evidence of LM-031 as a new NRF2 activator and its potential for treating SCA17 and other polyQ diseases. However, it should be noted that how LM-031/licochalcone A enhance NRF2 signaling remains to be investigated.  Oxidative Medicine and Cellular Longevity Using the TBP/Q 79 -GFP SH-SY5Y cell model, we showed that pCREB and CREB-mediated gene expression, BCL2 and GADD45B, were downregulated and BAX was upregulated in SCA17. CREB activation is pivotal to neuronal survival and knockdown of CREB leads to progressive neurodegeneration in the hippocampus and striatum, similar to the pathology seen in HD [59]. BCL2 is an antiapoptotic factor and its downregulation leads to decreased cell survival [60]. The knockdown of GADD45B decreases BCL2 and increases proapoptotic factor BAX, suggesting that GADD45B is an intrinsic neuroprotective molecule [61,62]. Neuronal apoptosis in SCA17 may be attributed to downregulated BCL2 and GADD45B and increased BAX in our study. This result is in consistence with previous reports that impaired CREB-dependent transcription plays an important role in the pathogenesis of polyQ diseases [7]. Several compounds or strategies activating CREB-dependent transcription have been shown to ameliorate pathology and phenotypes of cell and animal models of polyQ diseases including SCA3 and HD [11][12][13][14][15][16]63]. Valproic acid can protect against the toxicity of expanded ataxin-3 in an inducible cell model of SCA3 by upregulating CREB-dependent transcription through hyperacetylation [13]. Rolipram provides a neuroprotective effect to R6/2 HD mice via increasing the levels of activated CREB and of BDNF in the striatal spiny neurons [15]. More recently, doxycycline improves neurological deficits and reduces neuropathology in R6/2 mice by enhancing CREB activation in striatum as well as negatively modulating neuroinflammation [16]. However, the effects of CREBactivators have never been shown in SCA17 models. In this study, we demonstrate the neuroprotection effect of a new CREB activator on a SCA17 cell model and its potential for the treatment of polyQ diseases. Nevertheless, it is important to address the limitation of our study that the effects of the compounds tested were only demonstrated in cell models. Future studies using mouse models to show the neuroprotection in-vivo is warranted.  13 Oxidative Medicine and Cellular Longevity AICAR, an AMPK activator, raised pAMPKα and diminished the effects of aggregation reducing and neurite outgrowth enhancing by treatment with LM-031, which suggests increased pAMPKα contributing to neurotoxicity in SCA17 and LM-031 provides beneficial effect by reducing pAMPKα. It is noted that although licochalcone A reduced aggregates and promote neurite outgrowth, it did not attenuate pAMPKα, which suggests little effect of licochalcone A on AMPK pathway. Mitochondrial impairment and dysregulation of energy balance are involved in HD pathogenesis. Given that AMPK activation is important in response to energy deprivation, increased mitochondrial biogenesis increased by AMPK activation has been proposed as a potential therapeutic strategy [64][65][66]. However, studies led by us and other groups suggest that AMPK activation is associated with increased oxidative stress, which may be harmful to neurons and accelerate neurodegeneration [41,67]. Corrochano and colleagues have shown that endurance exercise is detrimental to HD via AMPK activation in skeletal muscle of HD mice [67]. Studies have shown that AMPK cascades are highly sensitive to oxidative stress [68][69][70], and a positive feedback interaction between AMPK activation and oxidative stress has been suggested in HD models [41]. In supporting the above implications, Ju et al. showed that an antioxidant, N-acetyl-L-cysteine, reduced the activation and nuclear enrichment of AMPKα1 and also increased the levels of BCL2, leading to rescue of neuronal death [41]. In accordance to these results, ours showed that LM-031 decreased aggregation, reduced ROS, and improved neurite outgrowth, which is mediated, at least partially, by reducing AMPKα phosphorylation. In contrast, cordycepin reduced aggregates and improved pathological abnormalities by enhancing autophagy, which is mediated through the activation of AMPK in two SCA3 mouse models [71]. Furthermore, activation of AMPK by A769662 and overexpression of an active form of AMPKα leads to improved cell viability in HD cell models [72]. Both studies suggest that AMPK activation may contribute to neuronal protection via a mechanism different from the AMPK-ROS pathway. Whether AMPK activation is beneficial or detrimental to polyQ diseases requires future studies to clarify further.
In order to know if the test compounds have chemical chaperone activity, we generated Trx-His-TBP/Q 20−61 proteins from IPTG-induced bacterial cells and performed thioflavin T binding and filter trap assays and the results showed evidence of the compounds' direct interference with aggregate formation (Figure 7). Whether the upregulated NRF2 and CREB and the downregulated pathway AMPK-ROS are the events subsequent to aggregate inhibition in the SCA17 SH-SY5Y cell model or directly affected by the compounds, or both, warrants future experiments to clarify further.

Conclusions
In the present study, we show that NRF2 and CREB pathways are compromised and AMPK is activated, which results in decreased BCL2 and GADD45B and elevated BAX and oxidative stress, and subsequent neurite outgrowth deficits as well as decreased neuronal survival in SCA17 (Figure 8). LM-031 exerts neuroprotective effects in SCA17 cell models by upregulating pCREB and NRF2 and reducing activated AMPKα ( Figure 8). Given that multiple pathogenic pathways are involved in polyQ including SCA17, LM-031 targeting multiple pathways to provide neuroprotection may have a significant perspective in drug development for SCA17. Given the lack of treatments that prevent disease progression in polyQ-mediated diseases, our study may shed light on the pathogenesis and therapeutic targets in SCA17 and other polyQ diseases. However, future studies in animal models are warranted to confirm our results before applying LM-031 to clinical trials.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.  14 Oxidative Medicine and Cellular Longevity