A Curcumin Derivative Activates TFEB and Protects Against Parkinsonian Neurotoxicity in Vitro

International Journal of Molecular Sciences

Article A Curcumin Derivative Activates TFEB and Protects Against Parkinsonian Neurotoxicity in Vitro

1,2, 1,2, 1 1,2 1,2 Ziying Wang †, Chuanbin Yang † , Jia Liu , Benjamin Chun-Kit Tong , Zhou Zhu , Sandeep Malampati 1, Sravan Gopalkrishnashetty Sreenivasmurthy 1, King-Ho Cheung 1,2, Ashok Iyaswamy 1, Chengfu Su 1 , Jiahong Lu 3 , Juxian Song 1,4,* and Min Li 1,2,* 1 Mr. & Mrs. Ko Chi-Ming Centre for Parkinson’s Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR 000000, China; [email protected] (Z.W.); [email protected] (C.Y.); [email protected] (J.L.); [email protected] (B.C.-K.T.); [email protected] (Z.Z.); [email protected] (S.M.); [email protected] (S.G.S.); [email protected] (K.-H.C.); [email protected] (A.I.); [email protected] (C.S.) 2 Institute for Research and Continuing Education, Hong Kong Baptist University, Shenzhen 518000, China 3 State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau SAR 000000, China; [email protected] 4 Medical College of Acupuncture-Moxibustion and Rehabilitation, Guangzhou University of Chinese Medicine, Guangzhou 510000, China * Correspondence: [email protected] (J.S.); [email protected] (M.L.) These authors contributed equally to this paper. † Received: 22 January 2020; Accepted: 19 February 2020; Published: 22 February 2020

Abstract: TFEB (transcription factor EB), which is a master regulator of autophagy and lysosome biogenesis, is considered to be a new therapeutic target for Parkinson’s disease (PD). However, only several small-molecule TFEB activators have been discovered and their neuroprotective effects in PD are unclear. In this study, a curcumin derivative, named E4, was identified as a potent TFEB activator. Compound E4 promoted the translocation of TFEB from cytoplasm into nucleus, accompanied by enhanced autophagy and lysosomal biogenesis. Moreover, TFEB knockdown effectively attenuated E4-induced autophagy and lysosomal biogenesis. Mechanistically, E4 induced TFEB activation is mainly through AKT-MTORC1 inhibition. In the PD cell models, E4 promoted the degradation of α-synuclein and protected against the cytotoxicity of MPP+ (1-methyl-4-phenylpyridinium ion) in neuronal cells. Overall, the TFEB activator E4 deserves further study in animal models of neurodegenerative diseases, including PD.

Keywords: TFEB; Parkinson’s disease; α-synuclein; curcumin derivatives; MTORC1

1. Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease in the world, affecting around 6.2 million people in 2015 [1]. The major hallmarks of PD are the loss of dopaminergic neurons (DAs) in the substantia nigra pars compacta (SNpc) and abnormal deposition of α-synuclein (α-syn) in different brain regions [2]. Unfortunately, the current drugs for PD only reduce motor symptoms while producing severe side effects [3,4]. Thus, neuroprotective or disease-modifying drugs that can slow down or stop the disease progression are urgently needed. Accumulating evidence has demonstrated that the impairment of the autophagy-lysosome pathway (ALP) plays an important role in the pathogenesis of PD. Therefore, stimulating ALP represents a promising strategy for PD treatment [5–8]. ALP, a cellular process for degrading misfolded cellular proteins and damaged organelles in lysosomes [6], has been proved to be tightly controlled by the transcription factor EB (TFEB) [9]. Several recent studies have investigated the roles and therapeutic

Int. J. Mol. Sci. 2020, 21, 1515; doi:10.3390/ijms21041515 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1515 2 of 14 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 14 andpotential therapeutic of TFEB potential in PD [10 of–14 TFEB]. Defective in PD ALP[10–14]. and Defective decreased ALP TFEB and activity decreased was found TFEB in activity postmortem was foundPD midbrains in postmortem [10]. The PD overexpression midbrains [10]. of TFEB The overexpression afforded protection of TF againstEB afforded both α-syn protection [10] and against MPTP both(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) α-syn [10] and MPTP (1-methyl-4-phenyl-1,2,3,6-t toxicityetrahydropyridine) [11] and improved toxicity motor [11] function and improved in the PD motoranimal function models. However,in the PD small-moleculeanimal models. activators However, of small-molecule TFEB with good activators brain bioavailability of TFEB with should good be braindiscovered bioavailability and evaluated should in be PD discovered animal models. and evaluated in PD animal models. Previously, we have identifiedidentified several TFEB activators from curcumin derivatives [15], [15], one of which directly binds to and activates TFEB without inhibiting the MTORC1 pathway and it exerts neuroprotective effects effects in animal models of Alzheime Alzheimer’sr’s disease [16]. [16]. In In this this study, study, we characterized another curcumin derivative, termed E4, as an an MT MTORC1-dependentORC1-dependent activator of TFEB. TFEB. We We found that compound E4 potentlypotently activatedactivated TFEB TFEB via via the the AKT-MTORC1 AKT-MTORC1 pathway, pathway, promoted promoted autophagy autophagy flux flux and andlysosomal lysosomal biogenesis, biogenesis, and and protected protected against againstα-syn α-syn and MPTPand MPTP toxicity toxicity in vitro. in vitro.

2. Results Results 2.1. Compound E4 Promoted the Nuclear Translocation of Endogenous and Exogenous TFEB in Vitro 2.1. Compound E4 Promoted the Nuclear Translocation of Endogenous and Exogenous TFEB in Vitro In normal conditions, TFEB locates in the cytoplasm. Being stimulated by various stresses, TFEB is In normal conditions, TFEB locates in the cytoplasm. Being stimulated by various stresses, TFEB de-phosphorylated and transported into the nucleus, where it binds to and activates multiple genes is de-phosphorylated and transported into the nucleus, where it binds to and activates multiple genes controlling autophagy and lysosomal biogenesis [17]. We monitored the localization of TFEB by controlling autophagy and lysosomal biogenesis [17]. We monitored the localization of TFEB by immunostaining to determine whether compound E4 (Figure1A) activates TFEB. E4 signiﬁcantly immunostaining to determine whether compound E4 (Figure 1A) activates TFEB. E4 significantly promoted the nuclear intensity of endogenous TFEB in N2a cells (Figure1B), and the stably promoted the nuclear intensity of endogenous TFEB in N2a cells (Figure 1B), and the stably overexpressed TFEB in HeLa cells (CF7) (Figure1C). The TFEB activator Torin 1 was used as a positive overexpressed TFEB in HeLa cells (CF7) (Figure 1C). The TFEB activator Torin 1 was used as a control [18]. Furthermore, the result from Western blots of cytosolic/nuclear fractions conﬁrmed that positive control [18]. Furthermore, the result from Western blots of cytosolic/nuclear fractions E4 activated TFEB (Figure1D,E). No cytotoxicity was observed for E4 at the tested concentrations confirmed that E4 activated TFEB (Figure 1 D–E). No cytotoxicity was observed for E4 at the tested (Figure S1). concentrations (Figure S1).

Figure 1. Cont.

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Figure 1.1.Compound Compound E4 promotedE4 promoted the nuclear the nuclear translocation translocation of endogenous of endogenous and exogenous and transcription exogenous transcriptionfactor EB (TFEB) factorin EB vitro (TFEB).(A) in Chemical vitro. (A) structure Chemical of structure E4. (B) of E4 E4. significantly (B) E4 significantly promoted promoted the nuclear the nuclearintensity intensity of endogenous of endogenous TFEB in TFEB N2a cellsin N2a compared cells compared to control to (0.1%control DMSO). (0.1% DMSO). After being After treated being µ withtreated E4 with (1 M) E4 for(1 μ 24M) h, for N2a 24 cells h, N2a were cells fixed were and fixed stained and with stained the anti-TFEBwith the anti-T antibodyFEB (red)antibody and DAPI(red) µ and(blue). DAPI Representative (blue). Representative images are images shown. are Scale shown. bar: 15 Scalem. bar: (C) E415μ significantlym. (C) E4 significantly promoted the promoted nuclear µ theintensity nuclear of intensity TFEB in CF-7of TFEB cells in compared CF-7 cells tocompared control (0.1%to control DMSO). (0.1% After DMSO). being After treated being with treated E4 (1 withM) E4and (1 positive μM) and control positive Torin1 control (250 Torin1 nM) for (250 24 h,nM) HeLa for cells24 h,stably HeLa expressingcells stably3XFlag-TFEB expressing 3XFlag-TFEB (CF-7) were fixed and stained with the anti-Flag antibody (green) and DAPI (blue). Representative images are (CF-7) were fixed and stained with the anti-Flag antibody (green) and DAPI (blue). Representative shown. Scale bar: 15 µm. Quantification of the number of cells with nuclear TFEB localization. Data are images are shown. Scale bar: 15μm. Quantification of the number of cells with nuclear TFEB presented as mean SEM of three replicates in a representative experiment. At least 100 cells were localization. Data are± presented as mean ± SEM of three replicates in a representative experiment. At analyzed in each treatment group. (D) The expression of Flag-TFEB in the cytosol and the nucleus least 100 cells were analyzed in each treatment group. (D) The expression of Flag-TFEB in the cytosol in CF-7 cells were detected by Western blotting after being treated with indicated compounds for and the nucleus in CF-7 cells were detected by Western blotting after being treated with indicated 6 h. (E) Relative intensity of TFEB is normalized to that of GAPDH and H3. Data are presented as compounds for 6 h. (E) Relative intensity of TFEB is normalized to that of GAPDH and H3. Data are mean SEM of three replicates in a representative experiment. * p < 0.05, ** p < 0.01, *** p < 0.001. presented± as mean ± SEM of three replicates in a representative experiment. * p < 0.05, ** p < 0.01, *** 2.2. Compoundp < 0.001. E4 Promoted Autophagy Flux and Lysosomal Biogenesis Depending on TFEB

2.2. CompoundWe next determined E4 Promoted whether Autophagy E4 Flux enhances and Lysosomal autophagy Biogenesis flux rather Depending than causing on TFEB lysosomal stress since lysosomal inhibition can activate TFEB [19]. First, we determined the effects of E4 on the autophagyWe next maker determined LC3B. E4 whether dose-dependently E4 enhances increased autophagy the flux level rather of LC3B-II than causing and the lysosomal number of stress LC3B sincepuncta lysosomal determined inhibition by Western can blotactivate (Figure TFEB2A) and[19]. immunostaining First, we determined (Figure the S2), effects respectively, of E4 inon N2a the autophagycells, which maker indicated LC3B. that E4 E4 dose-dependently increases autophagosomes increased in the cells. level Secondly, of LC3B-II in the and presence the number of a lysosome of LC3B punctainhibitor determined chloroquine by (CQ)Western [20], blot E4 further(Figure increased2A) and immunostaining the LC3B-II level (Figure (Figure S2),2B), respectively, indicating that in N2a E4 cells,promotes which the indicated formation that of autophagosomes. E4 increases autophagos Thirdly, inomes N2a in cells cells. that Secondly, were transfected in the withpresence a tandem of a lysosomefluorescent inhibitor mRFP-GFP-LC3 chloroquine (tfLC3) (CQ) plasmid [20], E4 [21 ],further E4 treatment increased significantly the LC3B-II increased level the(Figure red-only 2B), indicatingpuncta, whereas that E4 CQ promotes treatment the increased formation the yellowof autophagosomes. puncta (Figure 2Thirdly,D), thus in further N2a confirmingcells that were that transfectedE4 promoted with autophagy a tandem flux. Forfluorescent lysosome mRFP-GFP-LC3 biogenesis, we found (tfLC3) that plasmid E4 dramatically [21], E4 increased treatment the significantlylysosome contents, increased as determined the red-only by puncta, Lysotracker wher Redeas stainingCQ treatment (Figure increased2C), and increased the yellow the puncta levels (Figureof LAMP1 2D), and thus CTSD, further as confirming determined that by E4 Western promoted blot (Figureautophagy2E). flux. At the For gene lysosome level, E4biogenesis, expectedly we foundincreased that the E4 mRNA dramatically levels ofincreased several autophagy the lysoso andme contents, lysosome as genes determined that were by controlled Lysotracker by TFEB Red (Figurestaining2 F).(Figure 2C), and increased the levels of LAMP1 and CTSD, as determined by Western blot (Figure 2E). At the gene level, E4 expectedly increased the mRNA levels of several autophagy and lysosome genes that were controlled by TFEB (Figure 2F).

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Figure 2. Compound E4 promoted autophagy ﬂux and lysosomal biogenesis. (A) E4 dose-dependently increasedFigure the 2. levelCompound of LC3B-II E4 comparedpromoted autophagy with vehicle flux control and lysosomal (0.1% DMSO). biogenesis. The expression (A) E4 dose- of LC3-II in N2adependently cell after beingincreased treated the level with of di ﬀLC3B-IIerent concentration compared with of vehicle E4 for 24control h were (0.1% detected DMSO). by The Western blotting.expression Relative of LC3-II intensity in N2a of LC3B-IIcell after isbeing normalized treated with to thatdifferent of β -actinconcentration/ACTB. of Data E4 for are 24 presented h were as detected by Western blotting. Relative intensity of LC3B-II is normalized to that of β-actin/ACTB. mean SEM of three replicates in a representative experiment (B). The expression of LC3-II in N2a cell Data± are presented as mean ± SEM of three replicates in a representative experiment (B). The after being treated with indicated concentration of E4 and chloroquine (CQ) for 24 h were detected expression of LC3-II in N2a cell after being treated with indicated concentration of E4 and chloroquine β by Western(CQ) for blotting.24 h were detected Relative by intensity Western ofblotting. LC3B-II Relative is normalized intensity of toLC3B-II thatof is normalized-actin/ACTB. to that Data of are presented as mean SEM of 3 replicates in a representative experiment. (C) N2a cells were treated with β-actin/ACTB. Data± are presented as mean ± SEM of 3 replicates in a representative experiment. (C) vehicleN2a control cells were (0.1% treated DMSO), with E4vehicle (1 µM), control or CQ (0.1% (20 DMSO),µM) for E4 12 (1 h μ andM), thenor CQ stained (20 μM) with for 12 LysoTracker hours and Red DND-99then (50stained nM) with for 30LysoTracker minutes. Red DND-99 (50 nM) for 30 minutes. Fluorescence intensity of treated

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Fluorescence intensity of treated cells as measured by fluorescence microscopy. The numeric data are presented as means SEM from 3 independent experiments. (D) After the treated with vehicle control ± (0.1% DMSO), E4 (1 µM) or CQ (20 µM) in N2a cells that transfected with tf-LC3 plasmids for 16 h, the Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 14 fluorescence signal was captured by fluorescence microscopy and representative images are shown. Scale bar:cells 15 µasm. measured (E) E4 by treatment fluorescence increase microscopy. LAMP1, The numeric CTSD data levels are inpresented a dose-dependent as means ± SEM manner from as compared3 to independent vehicle control experiments. (0.1% ( DMSO).D) After the After treated N2a with cells vehicle treated control with (0.1% E4 andDMSO), positive E4 (1 μ controlM) or CQ Torin1 (250 nM) at(20 indicated μM) in N2a concentration cells that transfected for 24 h.with The tf-LC3 expressions plasmids for of LAMP1,16 h, the fluorescence pro-CTSD, signal mature-CTSD was captured by fluorescence microscopy and representative images are shown. Scale bar: 15 μm. (E) E4 were detected by Western blot assay and quantified. (F) CF-7 cells were treated with E4 (1 µM) for 16 h. treatment increase LAMP1, CTSD levels in a dose-dependent manner as compared to vehicle control mRNA transcript(0.1% DMSO). abundance After N2a was cells assessed treated bywith real-time E4 and positive PCR using control specific Torin1 primers(250 nM) for at indicated the indicated genes. Relative quantification is presented as means SEM of 3 independent experiments. * p < 0.05, ** concentration for 24 h. The expressions of LA±MP1, pro-CTSD, mature-CTSD were detected by p <0.01, ***Westernp < 0.001, blot ##assayp < and0.01. quantified. (F) CF-7 cells were treated with E4 (1 μM) for 16 h. mRNA transcript abundance was assessed by real-time PCR using specific primers for the indicated genes. Relative quantification is presented as means ± SEM of 3 independent experiments. * p < 0.05, ** p We knocked<0.01,down *** p < 0.001, TFEB ## p in< 0.01. N2a cells using Tfeb siRNA to determine whether TFEB activation contributes to the enhanced autophagy and lysosomal biogenesis induced by E4. Efficient knockdown (KD) of TFEBWe was knocked confirmed down TFEB by in Western N2a cells blotusing (FigureTfeb siRNA3A). to TFEBdetermine KD whether significantly TFEB activation abolished contributes to the enhanced autophagy and lysosomal biogenesis induced by E4. Efficient E4-induced increase in LC3B puncta and Lysotracker intensity (Figure3B,C). Meanwhile, E4-induced knockdown (KD) of TFEB was confirmed by Western blot (Figure 3A). TFEB KD significantly increase inabolished LAMP1 E4-induced and CTSD increase was in also LC3B significantly puncta and Lysotracker inhibited intensity in the (Figure TFEB 3 KD B–C). cells Meanwhile, (Figure 3D). Furthermore,E4-induced we confirmed increase in the LAMP1 roles and of TFEBCTSD was in E4-induced also significantly autophagy inhibitedin in HeLathe TFEB cells KD with cells TFEB knockout(Figure (KO). In3D). wild-type Furthermore, (WT) we cells, confirmed E4 significantly the roles of TFEB increased in E4-induced the level autophagy of LC3B-II in inHeLa the cells presence or absencewith of TFEB CQ. However,knockout (KO). no In significant wild-type (WT) change cells, in E4 LC3B-II significantly was increased observed the inlevel KO of cellsLC3B-II that in were treated withthe E4presence as compared or absence to of the CQ. control However, (Figure no signif3E).icant Together, change our in LC3B-II results was confirmed observed that in KO compound cells that were treated with E4 as compared to the control (Figure 3E). Together, our results confirmed that E4 promoted TFEB-mediated autophagy and lysosome biogenesis in vitro. compound E4 promoted TFEB-mediated autophagy and lysosome biogenesis in vitro.

Figure 3. TFEB is required for E4-induced autophagy ﬂux and lysosome biogenesis. (A) Transfected Figure 3. TFEB is required for E4-induced autophagy flux and lysosome biogenesis. (A) Transfected N2a cellsN2a with cells nontarget with nontarget siRNA siRNA (si-NT (si-NT, 50, nM)50 nM) and andTfeb TfebsiRNA siRNA ((siTfeb, ,50nm) 50 nm) for for48 h, 48 and h, the and the expressionexpression of TFEB of was TFEB evaluated was evaluated by western by western blotting. blotting. Relative intensity of TFEB are normalized to

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Relative intensity of TFEB are normalized to that of ACTB/β-actin. The data are presented as the mean SEM from 3 independent experiments. (B) N2a cells were transfected with nontarget siRNA (si-NT, ± 50 nM), Tfeb siRNA (siTfeb, 50 nM) for 48 h and treated with E4 (1 µM) for 16 h. N2a cells treated with E4 (1 µM) and stained with anti-LC3 antibody. The fluorescence of LC3 puncta was detected by fluorescence microscopy, representative images are shown. Scale bar: 15 µm. (C) N2a cells were transfected with nontarget siRNA (si-NT, 50 nM), Tfeb siRNA (siTfeb, 50 nM) for 48 h and treated with E4 (1 µM) for 16 h and then stained with LysoTracker Red DND-99 (50 nM) for 30 minutes. Representative images are shown. Scale bar: 15 µm. Fluorescence intensity of treated cells as measured by fluorescence microscopy. The numeric data are presented as means SEM from three independent experiments. ± (D) N2a cells were transfected with nontarget siRNA (si-NT, 50 nM), Tfeb siRNA (siTfeb, 50 nM) for 48 h and treated with E4 (1 µM) for 16 h. The expressions of LAMP1 and CTSD were examined by Western blotting. Representative blots are shown. Relative intensity of LAMP1 and CTSD are normalized to that of ACTB/β-actin. Data are presented as the mean SEM from 3 independent experiments. (E) Wild ± type Hela cells and TFEB knockout cells were treated with E4 (1 µM) in the presence or absence of CQ (20 µM) for 16 h. Representative blots are shown. Relative intensity of LC3B-II is normalized to that of GAPDH. The data are presented as the mean SEM from three independent experiments. * p < 0.05, ** ± p < 0.01, *** p < 0.001, ## p < 0.01, n.s. not significant. 2.3. Compound E4 Activates TFEB Via Inhibiting AKT-MTORC1 Pathway MTORC1 was shown to play a major role in the regulation of TFEB intracellular location [22]. MTORC1 is known to phosphorylate TFEB when nutrition is sufficient, and the inhibition of MTORC1 de-phosphorylates TFEB and promotes its nuclear translocation [23]. We examined the effects of E4 on the MTORC1 upstream kinase AKT, the phosphorylation of MTORC1, as well as substrate kinase P70S6K to determine whether E4 inhibits MTORC1 pathway to activate TFEB. E4 dose-dependently inhibited the phosphorylation of AKT and MTOR (Figure4A). E4 also inhibited p-MTOR and p-P70S6K time-dependently (Figure4B). Torin 1 was used as a positive control. We knocked down TSC2, a key upstream kinase of MTORC1, to continuously activate MTOR to further confirm the role of MTORC1 in E4-induced autophagy [24]. The results showed that E4 failed to inhibit p-P70S6K and increase LC3B-II in cells with TSC2 KD (Figure4C). The phosphorylation of TFEB at Ser142 is controlled by MTORC1 [23]. E4 treatment reduced the level of p-TFEB (Ser142) in cells that were transfected with non-target (NT) siRNA, but not in cells transfected with TSC2 siRNA (Figure4C). Furthermore, the colocalization of MTOR with LAMP1, which indicates the activation of MTORC1 [25], was abolished after E4 treatment (Figure4D). Overall, these results suggest that E4 activates TFEB via inhibiting the AKT-MTORC1 pathway. Int. J. Mol. Sci. 2020, 21, 1515 7 of 14 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 14

Figure 4. CompoundFigure 4. Compound E4 activates E4 activates TFEB TFEB via via inhibiting inhibiting AKT-MTORC1 AKT-MTORC1 pathway. (A pathway.) N2a cells were (A) treated N2a cells were treated withwith E4 E4 at at indicated indicated concentration concentration and positive and positive control Torin1 control (250 Torin1 nM) for (250 6 h. nM)Representative for 6 h. blots show the expression of phosphorylated (p-) and total AKT and mTOR. Data are presented as the mean Representative± SEM blotsfrom three show independent the expression experiments. of phosphorylated (B) N2a cells treated (p-) with and 1 μM total E4 for AKT different and durations mTOR. Data are (0–6 h). The expression of phosphorylated (p-) and total mTOR and p70S6K were examined by presented as the mean SEM from three independent experiments. (B) N2a cells treated with 1 µM E4 Western blotting.± Data are presented as the mean ± SEM from three independent experiments. (C) CF- for diﬀerent7 cells durations were transfected (0–6 h).with The nontarget expression siRNA (si-NT, of phosphorylated 25 nM), TSC2 siRNA (p-) (si-TSC2, and total 25 nM) mTOR for 48 and h p70S6K were examinedand treated by Western with E4 blotting. (1 μM) Datafor 24 areh. presentedRepresentative as theblots mean show theSEM expression from three of TSC2, independent ± experiments.phosphorylated (C) CF-7 (p-) cells and were total mTOR, transfected p-TFEB with(Ser142) nontarget and LC3 proteins. siRNA The (si-NT, relative 25 intensity nM), of TSC2 p- siRNA p70S6K is normalized to that of p70S6K. Relative intensity of p-TFEB and LC3-II is normalized to that (si-TSC2, 25 nM) for 48 h and treated with E4 (1 µM) for 24 h. Representative blots show the expression of DAPDH. (D) N2a cells were transfected with mTOR and RFP-LAMP1 plasmids for 48 h and then treated of TSC2, phosphorylatedwith E4 (1 μM) for (p-)24 h. and The cells total were mTOR, stained p-TFEB with anti-mTOR (Ser142) antibody. and LC3 Fluorescence proteins. Thewas captured relative intensity of p-p70S6Kby isfluorescence normalized microscopy to that and of p70S6K.representa Relativetive images intensity are shown. of Scale p-TFEB bar: 25 and μm. LC3-II * p < 0.05, is normalized** p < to that of DAPDH.0.01. (D) N2a cells were transfected with mTOR and RFP-LAMP1 plasmids for 48 h and then treated with E4 (1 µM) for 24 h. The cells were stained with anti-mTOR antibody. Fluorescence was

captured by ﬂuorescence microscopy and representative images are shown. Scale bar: 25 µm. * p < 0.05, ** p < 0.01. 2.4. Compound E4 Reduces α-syn Levels and Protects Against MPP+ Toxicity in Vitro TFEB activation has been shown to be protective in the cellular and animal models of PD [10–14]. Therefore, we primarily tested whether compound E4 exerts neuroprotection in cellular models of PD. In N2a cells transfected with A53T α-syn plasmid, E4 treatment dose-dependently reduced the level of overexpressed α-syn (Figure5A). The MPP + (1-methyl-4-phenylpyridinium ion) is the most common neurotoxin that is used for modeling PD, since it mimics the slow neuronal cell death of PD [26]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 14

2.4. Compound E4 Reduces α-syn Levels and Protects Against MPP+ Toxicity in Vitro TFEB activation has been shown to be protective in the cellular and animal models of PD [10- 14]. Therefore, we primarily tested whether compound E4 exerts neuroprotection in cellular models of PD. In N2a cells transfected with A53T α-syn plasmid, E4 treatment dose-dependently reduced the level of overexpressed α-syn (Figure 5A). The MPP+ (1-methyl-4-phenylpyridinium ion) is the most common neurotoxin that is used for modeling PD, since it mimics the slow neuronal cell death of PD Int.[26]. J. Mol.We Sci.pre-treated2020, 21, 1515 PC12 cells with E4 for six hours and then co-treated cells with MPP+ for 48 hours.8 of 14 The cell viability results showed that E4 significantly reduced cell death and dose-dependently Weprotected pre-treated PC12 PC12 cells cellsagainst with MPP E4 for+-induced six hours cytotoxicity and then co-treated(Figure 5 B cells–C). withCell MPPviability+ for was 48 h.measured The cell viabilityby Alamar results blue showedassay. Interestingly, that E4 significantly this neuroprotective reduced cell effect death was and abolished dose-dependently by co-treating protected cells PC12with cellslysosome against inhibitor MPP+-induced CQ, suggesting cytotoxicity that (Figure the neuroprotective5B,C). Cell viability effects was of measured E4 are through by Alamar the blueautophagy-lysosome assay. Interestingly, pathway this neuroprotective (Figure 5 B–C). e ffTakenect was together, abolished E4 byhas co-treating shown good cells neuroprotective with lysosome inhibitoreffects in CQ,two suggestingdifferent PD that cell the models. neuroprotective effects of E4 are through the autophagy-lysosome pathway (Figure5B,C). Taken together, E4 has shown good neuroprotective e ffects in two different PD cell models.

Figure 5. Compound E4 reduces α-syn levels and protects against MPP+ toxicity in vitro.(A) N2a cellsFigure were 5. Compound transfected E4 with reduces A53T αα-syn-syn levels plasmid and and protects then treatedagainst withMPP+ E4 toxicity at indicated in vitro. concentration. (A) N2a cells Datawere aretransfected presented with as A53T the mean α-syn plasmidSEM from and 3 then independent treated with experiments. E4 at indicated (B) Bright concentration. field of PC12 Data ± cellsare presented after incubation as the mean with E4± SEM (1 µ M)from present 3 independent or absence experiments. of CQ (20 µ (M)B) inBright MPP field+ (1 mM)of PC12 treated cells cells.after Scaleincubation bar: 50 withµm. E4 (C (1)μ CellM) viabilitypresent or of absence E4 present of CQ or absence(20μM) in of MPP CQ against+ (1mM) MPP treated+ induced cells. Scale cell injury.bar: 50 Dataμm. ( areC) Cell presented viability as theof E4 mean presentSEM or fromabsence three of independentCQ against MPP experiments.+ induced* cellp < injury.0.05, ** Datap < 0.01, are ± #presentedp < 0.05, n.s.as the not mean significant. ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, # p < 0.05, n.s. not significant. 3. Discussion 3. DiscussionIn this study, we discovered a novel TFEB activator, named E4, from synthesized curcumin derivatives.In this study, By activating we discovered TFEB, E4 a promoted novel TFEB autophagy activator, flux named and lysosomalE4, from synthesized biogenesis, whichcurcumin are essentialderivatives. for theBy activating clearance ofTFEB, protein E4 aggregates,promoted autophagy as seen in neurodegenerativeflux and lysosomal diseases, biogenesis, including which PDare (Figureessential6). for the clearance of protein aggregates, as seen in neurodegenerative diseases, including PD (FigureThe 6). activation of TFEB is mainly controlled by its phosphorylation. TFEB is phosphorylated by several regulators, such as GSK3B, AKT, MTORC1, and ERK2, and it remains inactive in the cytoplasm [18]. MTORC1 is one of the most important regulators for TFEB by controlling its phosphorylation [27]. The phosphorylation site of Ser142 is regarded as an essential site for MTORC1 to regulate TFEB at the surface of lysosome membrane. So far, our data revealed that AKT-MTORC1 is involved in E4-induced TFEB activation. However, whether E4 acts on other upstreaming regulators of TFEB needs to be fully evaluated.

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Figure 6. Underlying mechanisms by which compound E4 activate TFEB and autophagy-lysosome Figure 6. Underlying mechanisms by which compound E4 activate TFEB and autophagy-lysosome process. Compound E4 inhibited AKT as well as MTORC1 pathway and further promoted the nuclear process.accumulation Compound ofE4 TFEB. inhibited Cytoplasmic AKT as TFEB well isas highly MTORC1 phosphorylated pathway and locatedfurther in promoted the surface the of nuclear accumulationlysosome of whichTFEB. bind Cytoplasmi with MTOR.c CompoundTFEB is highly E4 inhibited phosphorylated the MTOC1 activity and andlocated promoted in the TFEB surface of lysosomedephosphorylation. which bind with Dephosphorylated MTOR. Compound TFEB translocatedE4 inhibited into the nucleus MTOC1 and activity bind to autophagy and promoted and TFEB dephosphorylation.lysosome related Dephosphorylat genes site. Eventually,ed TFEB compound translocated E4 promoted into TFEB nucl fromeus cytoplasm and bind to nucleusto autophagy and and induced autophagy and lysosome biogenesis. lysosome related genes site. Eventually, compound E4 promoted TFEB from cytoplasm to nucleus and inducedTo date, autophagy there is and no cure lysosome for PD. biogenesis. New pathological mechanisms and new drug targets are extensively explored. The impairment of ALP in PD has been increasingly recognized and new Thedrugs activation promoting of TFEB ALP are is beingmainly discovered controlled and by evaluated its phosphorylation. in PD models [6,7 TFEB]. Since is TFEB phosphorylated controls by the whole ALP process, it presents an ideal target for PD drug development [12]. Major cell several regulators, such as GSK3B, AKT, MTORC1, and ERK2, and it remains inactive in the cytoplasm [18]. MTORC1 is one of the most important regulators for TFEB by controlling its phosphorylation [27]. The phosphorylation site of Ser142 is regarded as an essential site for MTORC1 to regulate TFEB at the surface of lysosome membrane. So far, our data revealed that AKT-MTORC1 is involved in E4-induced TFEB activation. However, whether E4 acts on other upstreaming regulators of TFEB needs to be fully evaluated. To date, there is no cure for PD. New pathological mechanisms and new drug targets are extensively explored. The impairment of ALP in PD has been increasingly recognized and new drugs promoting ALP are being discovered and evaluated in PD models [6,7]. Since TFEB controls the whole ALP process, it presents an ideal target for PD drug development [12]. Major cell models of PD are α-syn overexpression model and the neurotoxins induced cytotoxicity model. Recent studies

Int. J. Mol. Sci. 2020, 21, 1515 10 of 14 models of PD are α-syn overexpression model and the neurotoxins induced cytotoxicity model. Recent studies indicated that MPP+ impaired the autophagy flux and induced α-syn aggregation [28–30]. Additionally, MPP+ treatment was also found to decrease the LAMP1 level in human dopaminergic BE-M17 neuroblastoma cells, as well as MPTP, treated mice brain [31]. Consequently, the MPP+ cell model might be an appropriate model for studying the protection of PD. The aggregation of α-syn has been reported to block autophagy flux, whereas enhanced autophagy might promote α-syn degradation [28,32]. Our preliminary data showed that the protective effects of E4 in these cell models. However, whether E4 has good brain bioavailability and, therefore, ideal neuroprotective efficacy in PD animal models needs to be further investigated.

4. Material and Methods

4.1. Materials The analog of curcumin E4 was synthesized according to our previous study and stored in DMSO (Stock concentration 1 mM) at 35 C. MPP+ (1-Methyl-4-phenylpyridinium iodide) (D048) and − ◦ anti-Flag (F1804) antibody were purchased from Sigma-Aldrich. (St. Louis, MO, USA) TFEB siRNA (L-050607-02-0005), TSC2 siRNA (L-003029-00-0005), and non-target siRNA were purchased from Dharmacon. Torin1 (2273-5) was purchased from BioVision Inc. Anti-β-actin/ACTB (sc-488 47778) and anti-α-synuclein (c20) (J1615) were purchased from Santa Cruz Biotechnology. Anti-LAMP1 (AB24170) and anti-CSTD (AB75852) antibody were purchased from Abcam Company (Abcam, Cambridge, UK). Anti-phospho-P70S6K (Thr389) (9234), anti-P70S6K/RPS6KB1 (9202), anti- H3F3A/histone H3 (D1H2), anti-phospho-mTOR (Ser2448) (2971S), anti-mTOR (2972S), anti-α-synuclein (2628S), anti-TSC2 (4308s), anti-AKT (9272S), and anti- phosphor-AKT (Ser473) (9276S) antibodies were purchased from Cell Signaling Technology company. Anit-TFEB (A301-852A) antibody was purchased from Bethyl Laboratories, Inc. Anti-TFEB (Ser142) (Q3004775) was purchased from Millipore Company. Anti-GAPDH (GTX100118) antibody was purchased from GeneTex Company. Anti- LC3B (NB100-2220) was purchased from Novus Biologicals. LysoTracker®Red DND-99 (L-7528), DMEM (11965-126), FBS (10270-106), Opti-MEM I (31985-070), and alamarBlue™ Cell Viability Reagent (DAL1025) were purchased from Life Technologies. TRIzol™ Reagent (15596018) was purchased from Thermo Fisher Scientiﬁc Inc. PrimeScript™ RT Master Mix (Perfect Real Time) (RR036A) was purchased from Takara Bio Company. Alexa Fluor®488 goat anti-mouse IgG (A-11001), Alexa Fluor®488 goat anti-Rabbit IgG (A-11034), and Alexa Fluor 488 (A-11008) were purchased from Life Technologies (Carlsbad, CA, USA).

4.2. Cell Culture and Drug Treatment N2a cells, PC12 cells, wild type Hela cells, and TFEB knock out Hela cells were cultured in DMEM supplemented with 10% FBS. Wild type Hela cells and TFEB knock out Hela cells were the gift from Prof. Richard J. Youle (National Institute of Neurological Disorders and Stroke) and Prof. Myung-Shik Lee (Yonsei University College of Medicine). The HeLa cells stably expressing 3x-Flag-TFEB (CF-7) cells were maintained in DMEM that was supplemented with 10% FBS and 50 µg/mL G418 [27]. All of the cells were maintained with cell culture medium containing 100 units/mL penicillin/streptomycin mixture (Invitrogen, Carlsbad, CA, USA) at 37 ◦C, gassed with 5% CO2. For drug treatment, the full medium was replaced by fresh Opti-MEM I containing the compounds (in 0.1% DMSO) and then incubated for the indicated time periods.

4.3. Cell Transfection and Gene Knockdown Assay For overexpression, the cells were transfected by using Lipofectamine 3000 (L3000015) following the manufacturer’s instruction. For gene knockdown, the cells were transfected with siTFEB or siTSC2 and the nontarget siRNA by using Lipofectamine RNAiMAX (Invitrogen, 13778030) and incubated at 37 ◦C for 48 h. The cells were treated with indicated drugs after being transfected with siRNAs and plasmids for 48–72 h. Int. J. Mol. Sci. 2020, 21, 1515 11 of 14

4.4. Quantitative Real-Time PCR Total RNA was extracted from cells while using the TRIzol™ Reagent (15596018). Reverse transcription was performed using PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio Inc, Shiga, Japan, RR047A). Autophagy and lysosome gene primers were retrieved from Origene and from a previous study [10] and synthesized by Life Technologies. Table S1 lists the oligonucleotide sequences. Real-time PCR was carried out with the PrimeScript™ RT Master Mix (Perfect Real Time) (RR036A) using the ViiA™ 7 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). Fold changes were calculated while using the ∆∆CT method, and the results were normalized against an internal control (GAPDH).

4.5. Alamar Blue Assay Add 1 of 10 volume of alamarBlue™ Cell Viability Reagent (DAL1025, Life Technologies) directly to cells in the culture medium, incubate for one to four hours at 37 ◦C in a cell culture incubator, being protected from direct light. Monitor the absorbance of Alamar Blue at 570 nm, using 600 nm as a reference wavelength. Results of treatment were read relative to controls, while assuming the absorbance of controls was 100%.

4.6. Isolation of the Cytosol and the Nucleus Fractions Cytosol and nucleus extracts were prepared according to a previous protocol. In brief, at the end of drug treatment, the cells were washed with ice-cold PBS, centrifuged, and then re-suspended in cold lysis buffer containing 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA), 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 0.5 mg/mL benzamidine, 0.1 mg/mL leupeptin, and 1.2 mg/mL aprotinin. The cells were allowed to swell on ice for 15 min. NP-40 (10% (v/v)) was subsequently added to the cell suspensions. The samples were vigorously vortexed for 10 s. The homogenates were centrifuged for 50 s at 16,000 g, and the supernatant was used as cytosolic extract. The nuclear pellet × was re-suspended in cold extraction buffer containing 20 mM HEPES, pH 8.0, 1 mM EDTA, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 0.5 mg/mL benzamidine, 0.1 mg/mL leupeptin, 1.2 mg/mL aprotinin, and 20% glycerol. All of the protein fractions were stored at 30 C until use. − ◦ 4.7. Western Blot The cells were washed twice with cold 1x PBS (phosphate-buffered saline) and then lysed on ice with 1 RIPA Lysis buffer (9803, Life Technologies) with complete protease inhibitor mixture (04693124001, × Roche Applied Science, Penzberg, Germany) and phosphatase inhibitor (B15001, Bio-connect BV, Huissen, Netherlands). The protein concentrations were determined by Bio-Rad Bradford assays while using bovine serum albumin (BSA) as standard. Proteins were separated by 10–15% SDS-PAGE, transferred, and blotted with the antibodies described. The blots were then incubated with primary and secondary antibodies. The protein signals were detected by the ECL kit (Pierce, 32106) and quantified while using ImageJ software (Version 1.52, NIH, USA).

4.8. Immunocytochemistry The cells were seeded on coverslips placed in 24-well plates. For autophagy flux assessment, N2a cells were transfected with tfLC3 plasmid for 24 h and then treated with the indicated drugs. For lysosome activity was evaluated using LysoTracker Red DND99 (L7528, Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s instructions. During the last 1 h of drug treatment, 50nM LysoTracker Red DND99 added to plates. After treatment, the slices were washed three times with PBS and fixed with 4% paraformaldehyde, permeabilized in 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA, T8787) and blocked with 3% BSA. After blocking, slides were Int. J. Mol. Sci. 2020, 21, 1515 12 of 14

stained with anti-Flag (1:600), anti-TFEB (1:200), and anti-LC3B (1:500) antibodies overnight at 4 ◦C. Alexa Fluor®488 (green) secondary antibodies (1:1000) were added for 1 h at room temperature. After nuclear staining with DAPI, the slices were mounted with FluorSave reagent (Calbiochem, Darmstadt, Germany, 345789). The cells were visualized while using the API DeltaVision Personal Imaging System and Confocal Laser Scanning Microscope (Leica TCS SP8, Wetzlar, Germany).

4.9. Statistical Analysis Each experiment was performed at least three times, and the results were presented as mean SEM. ± Student’s t-test or One-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test while using GraphPad Prism 7.0 software (San Diego, CA, USA) packages. A probability value of p < 0.05 was considered to be statistically signiﬁcant.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/4/1515/s1. Author Contributions: Conceptualization, J.S. and M.L.; Data curation, Z.W.; Formal analysis, Z.W., C.Y. and S.G.S.; Funding acquisition, C.Y. and M.L.; Methodology, J.L. (Jia Liu), S.M. and A.I.; Project administration, C.Y., J.L. (Jiahong Lu) and M.L.; Resources, K.-H.C.; Software, B.C.-K.T., Z.Z. and C.S.; Writing—original draft, Z.W., C.Y.; Writing—review & editing, Z.W., C.Y., J.S., M.L. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by Shenzhen Science and Technology Innovation Commission (SZSTI, 201803023000787), (JCYJ, 20180302174028790) and (JCYJ20180507184656626). the National Natural Science Foundation of China (81703487, 81773926), the General Research Fund (GRF/HKBU12101417, GRF/HKBU12100618), and the Hong Kong Health and Medical Research Fund (HMRF14150811, HMRF15163481) from Hong Kong Government, and research funds (HKBU/RC-IRCs/17-18/03, HKBU/RC-IRMS/15-16/04, FRGI/17-18/041, FRGII/17-18/021) from Hong Kong Baptist University. Acknowledgments: We would like to thank Richard J. Youle (National Institute of Neurological Disorders and Stroke) and Myung-Shik Lee (Yonsei University College of Medicine) for providing TFEB knock out Hela cells. We thank Tamotsu Yoshimori (Osaka University) for providing the tfLC3 plasmid. We would like to thank for Carol Chu’s support and assistant. We would like to thank Martha Dahlen for her English editing. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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