Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disease, clinically characterized by motor abnormalities (rest tremor, rigidity, bradykinesia, shuffling gait, and postural instability) and non-motor symptoms (depression, anxiety, dementia, sleep disorder)1. This characteristic movement restriction reduces the ability of PD patients to work, and lessens their quality of life. Indeed, late-stage PD patients often cannot take care of themselves, which significantly burdens individual caretakers, families, and society2. The loss of dopaminergic neurons (DAs) in the substantia nigra pars compacta (SNpc) and the presence of abnormal α-synuclein (α-syn) aggregates (Lewy bodies) are the principal pathological hallmarks of PD3. Misfolded and aggregated α-syn may lead to different pathogenic effects, including induction of oxidative stress4, increase of membrane permeability5, interruption of axonal transport6, synaptic dysfunction7, mitochondrial dysfunction44. A natural product, harmol has fewer side effects and plays an important role in health maintenance and disease control. Finally, natural products can be used as lead compounds for structural modification to synthesize derivatives.

Traditional animal models of PD are designed using toxins, such as 6-hydroxydopamine (6-OHDA), rotenone, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Although toxin-treated models cause the dysfunction and death of dopaminergic neurons within substantia nigra, one of the major drawbacks of traditional models is the lack of Lewy body pathology45. A53T α-syn transgenic mice exhibit a full range of pathology including α-syn aggregation, oligomers, fibrils, phosphorylation, ubiquitination, and progressive neurodegeneration45. In this study, A53T α-syn transgenic animals developed the α-syn inclusions widely distributed throughout each brain region (Fig. 6), and the transgenic mice showed serious motor impairment (Fig. 5). In addition, the internalization and aggregation of misfolded α-syn can lead to ALP dysfunction in cellular and animal models or vice versa, suggesting that a crosstalk between α-syn aggregation and autophagic/lysosomal dysfunction may exist46. Our results prove that the overexpression of α-syn suppresses autophagic/lysosomal function (Figs. 3, 4, 6, and 7). However, the α-syn load did not affect the expression of dopaminergic neuron marker tyrosine hydroxylase (TH) within substantia nigra and striatum (Supplementary Fig. 2). Meanwhile, HPLC (high performance liquid chromatography) analysis showed no significant difference in total striatal DA, HVA (homovanillic acid), and DOPAC (3, 4-dihydroxyphenylacetic acid) content between wild-type and A53T α-syn transgenic mice (Supplementary Fig. 3). Increasing evidence suggests that mice overexpressing A53T α-syn do not lose dopaminergic neurons in substantia nigra, and motor deficits are caused by a loss of brain stem neurons and anterior horn motor neurons of the spinal cord45. In A53T α-syn transgenic mice, dopaminergic neurons do not show the same selective vulnerability that humans demonstrate. Why these neurons have different cross-species characteristics remains unclear, but the lack of neuromelanin formation in mice may be a determinant31. In addition, murine dopaminergic neurons display inexplicable resistance to α-syn-induced neurotoxicity compared with other neuronal populations47.

Autophagy and lysosome biogenesis could be enhanced by activating TFEB in vitro and in vivo48. Overexpressing TFEB or inducing its nuclear translocation stimulates ALP functioning and attenuates α-synuclein pathology19. In this study, we identified a natural ALP enhancer, harmol, that promotes the clearance of α-syn in vitro and in vivo (Figs. 1 and 6), and harmol-activated TFEB induces ALP (Figs. 2–4 and 7). AMPK promotes autophagy by inhibiting mTOR in the cytoplasm to allow nuclear translocation of TFEB49. Activation of AMPK-mTOR-TFEB axis-mediated autophagy promotes the clearance of toxic protein aggregates. Indeed, harmol administration increases phosphorylation of AMPK, inhibits phosphorylation of mTOR, and increases the expression of TFEB (Figs. 4 and 7). Furthermore, AMPK has been reported as an activator of autophagy via inhibition of the mTOR complex and direct phosphorylation and activation of ULK1. Specifically, AMPK can promote autophagy by directly activating ULK1 via phosphorylation of Ser317, Ser555, and Ser77750, while mTOR inhibits this process through ULK1 phosphorylation at Ser757. In this study, the p-ULK1(Ser757)/ULK1 is down-regulated, while p-ULK1(Ser317)/ULK1 and p-ULK1(Ser555)/ULK1 are not significantly affected (Figs. 3 and 7). This may indicate that AMPK activation mainly involves mTOR-ULK1 (Ser757) pathway in this study, which regulates autophagy. Compound C/Dorsomorphin is a primary reagent used as an AMPK inhibitor. Compound C can bind to phosphorylated-state mimic T172D mutant kinase domain of the human AMPKα subunit, and compound C can also block the AICAR cellular uptake by competing for adenosine transporter-binding sites51. Inhibition of the AMPK-mTOR-TFEB signaling pathway using AMPK blocker compound C diminishes the effects of harmol on autophagy and α-syn clearance (Fig. 4c–g). Hence, these studies suggest that harmol promotes α-syn clearance via the AMPK-mTOR-TFEB signaling pathway (Fig. 8).

Fig. 8: Underlying mechanisms by which harmol degrades α-syn.
figure 8

Harmol activates the AMPK and inhibits the mTOR. Cytoplasmic TFEB is highly phosphorylated and located in the lysosomal membrane, which binds with mTOR. Harmol inhibits the mTOR activity and promotes the dephosphorylation of TFEB. Dephosphorylated TFEB translocates into the nucleus, where it binds to the CLEAR sequence, leading to upregulation of the autophagic and lysosomal genes. Eventually, harmol enhances the autophagy-lysosome process and promotes the degradation of α-syn.

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Collectively, our research confirms that harmol activates the AMPK-mTOR-TFEB mediated ALP pathway in vitro and in vivo, contributing to the degradation of pathogenic proteins, the restoration of autophagic flux, and lysosomal biogenesis, and the improvement of motor impairment. These results increase the possibility that harmol may be developed into a therapeutic agent against aggregate-prone protein-associated neurodegenerative diseases such as PD, but there are still deficiencies and limitations. It is still not clear how harmol induces AMPK phosphorylation, and future work should aim to identify the upstream molecular signal or target for AMPK activation. Several studies have shown that TFEB also activates AMPK and, in turn, establishes a unique feed-forward loop between TFEB and AMPK52. Thus, harmol may promote autophagy via the APMK-mTOR-TFEB feed-forward loop, and future studies should test this possibility, although harmol may utilize other pathways to degrade α-syn or other mechanisms to regulate ALP.

Methods

Reagents and antibodies

Harmol was purchased from Tokyo Chemical Industry. Doxycycline was purchased from MedChemExpress. Dorsomorphin dihydrochloride was purchased from Targetmol. G418 sulfate was purchased from Amresco. Dopamine hydrochloride, homovanillic acid, 3, 4-dihydroxyphenylacetic acid, tri-sodium citrate dihydrate, citric acid, chloroquine, anti-Flag M2, and anti-α-synuclein antibodies were purchased from Sigma-Aldrich. Anti-TFEB antibody was purchased from Proteintech. Anti-tyrosine hydroxylase, anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-ULK1 (Ser757), anti-phospho-ULK1 (Ser317), anti-phospho-ULK1 (Ser555), anti-ULK1, anti-phospho-AMPKa (Thr172), anti-AMPKa, anti-LAMP1, anti-SQSTM1/p62, anti-TFEB, and anti-CTSD/cathepsin D antibodies were purchased from Cell Signaling Technology. Anti-phospho-α-synuclein and anti-LC3B antibodies were purchased from Abcam. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was purchased from Beyotime Biotechnology.

Animals and treatments

Male hA53T α-syn transgenic mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Animals were propagated and fed at the Technology Industrial Park Development Co., Ltd of Guangzhou University of Chinese Medicine. The mice were housed under a 12 h light-dark cycle at 23 ± 2 °C and 60 ± 15% relative humidity. All animal care and experimental procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Technology Industrial Park Development Co., Ltd of Guangzhou University of Chinese Medicine, and approved by the committee.

hA53T mice were randomly divided into four groups: saline (n = 6), harmol 10 mg/kg (n = 6), harmol 20 mg/kg (n = 6) and harmol 40 mg/kg (n = 6). Ten-month-old hA53T α-syn transgenic mice were treated with harmol (oral gavage twice daily) or an equal volume of saline for 1 month. Wild-type mice (n = 10) were administrated saline.

Behavioral tests

To evaluate α-syn-induced behavioral deficits, the pole test, rotarod test, open-field test, and automated treadmill gait test was executed. All behavioral studies were double-blinded and repeated three times to obtain the average value for statistical analysis.

Pole test

The climbing-pole test device was a metal rod that was ~50 cm high and 1 cm in diameter, wrapped with gauze to increase friction53. The mice were placed on top of the vertical pole facing down, and the descent time from top to bottom was recorded. Before the test, each mouse was trained for three consecutive days. The experiment was repeated three times in a row on the same day, and the average climbing time was calculated as the evaluation value.

Rotarod test

For the rotarod test, the mice were trained for 3 consecutive days before the actual test54. Mice were placed on an accelerating rotarod cylinder, and the latency time was tested. The speed was slowly accelerated from 4 to 40 rpm within 4 min. The data are presented as the average latency time (three independent trials) on the rotarod.

Open-field test

Animals were placed individually into the center of an acrylic apparatus (50 cm × 50 cm × 40 cm) and allowed to freely explore for 5 min under dim light. The apparatus was cleaned with 70% ethanol solution between each trial to avoid olfactory cues between animals. A video tracking system was used to record the total distance traveled as a measure of autonomous movement55.

Automated treadmill gait test

Treadmill gait assessment was performed with the DigiGait imaging apparatus (Mouse Specifics Inc.)56. Mice were placed on a motorized treadmill within a plexiglass compartment (~25 cm long and 5 cm wide). A camera mounted under the transparent treadmill belt acquired digital video images at 80 frames per second to visualize paw contacts. The treadmill was set at a fixed speed of 15 cm/sec so that most animals could move continuously. Digital videos were analyzed by DigiGait software, which automatically recognized the pawprints, and if necessary, manual changes in the images were made to properly distinguish the pawprints from the background. Then, the images were automatically processed by the software to calculate the values of various gait parameters, including stride frequency, stride width, step angle, absolute paw angle, swing, stride, and stance.

HPLC-ECD analysis

Striatum was homogenized on ice with 0.1 M perchloric acid. After centrifugation at 12,000 × g for 15 min, the supernatant was analyzed using an Agilent HPLC-ECD system (Agilent Technologies Inc., USA) with Agilent Eclipse plus C18 column (4.6 × 100 mm, 3.5 μm). The mobile phase was methanol (10%) and aqueous solution (90%, containing 13.764 g/L citric acid, 10.147 g/L tri-sodium citrate dihydrate, 269.25 mg/L sodium octane sulfonate, 37.2 mg/L ethylenediaminetetraacetic acid disodium salt dehydrate and 149.2 mg/L potassium chloride), and detection was carried out at 35 °C with a flow rate of 0.8 mL/min. For ECD, the detecting voltage was maintained at 750 mV, and the response range of the detector was set at 50 nA. The retention time of 3, 4-dihydroxyphenylacetic acid (DOPAC), dopamine (DA) and homovanillic acid (HVA) was 4.5, 6.2 and 10.3 min, respectively.

Cell culture and drug treatment

N2a cells were provided by Prof. Simon MingYuen Lee (State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, China) as a gift, and the cells were maintained in DMEM supplemented with 10% FBS. HeLa cells stably expressing 3xFlag-TFEB were supplied by Prof. Ju-**an Song (Guangzhou University of Chinese Medicine, Guangzhou, China) as a gift, and cultured in DMEM containing 10% FBS and 500 μg/mL G418. Inducible PC12 cells were generously gifted from professor Yadong Huang (**an University, Guangzhou, China) and maintained in DMEM supplemented with 5% HS, 10% FBS, and 200 µg/mL G418. For drug treatment, the whole medium was replaced with a fresh medium containing the compounds and then incubated for the indicated periods.

Cell viability

Cells were plated in a 96-well plate for 24 h. After 24 h of compound treatment, cell cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis, following the manufacturer’s guidelines. Absorbance was measured spectrophotometrically at 570 nm with a microplate reader (Tecan, Switzerland).

High-content screening assay

To quantify TFEB subcellular localization, a high-content assay was performed using HeLa cells stably expressing 3xFlag-TFEB and N2a cells. Cells were seeded in 96-well plates for 24 h and treated with different concentrations of compounds. After 24 h, cells were fixed, permeabilized, blocked, and stained with anti-Flag M2 or anti-TFEB overnight at 4 °C. DAPI, Alexa Fluor®488 (green), or Alexa Fluor® 594 (red) secondary antibodies were added for 2 h at room temperature. Images were acquired by confocal automated microscopy (IN Cell Analyzer 6000, USA). The ratio value of the average nuclear fluorescence intensity divided by the average cytosolic fluorescence intensity was calculated57. Data are represented by the percentage of nuclear translocation at different concentrations of each compound using GraphPad Prism 7 software (GraphPad Software, USA).

Autophagy flux assay

N2a cells were transfected with mCherry-GFP-LC3B adenovirus for 24 h, and then the medium was replaced with a regular culture medium. After 48 h, the cells were respectively treated with harmol, CQ and rapamycin for 2 h. Images were obtained using a fluorescent confocal microscope (Zeiss, Germany) and were quantified by ImageJ software (Version 1.52, USA).

Lyso-Tracker Red staining

Lysosome activity was evaluated using Lyso-Tracker Red (Beyotime Biotechnology, C1046) according to the manufacturer’s protocol. After 24 h of drug treatment, Lyso-Tracker Red (50 nM) was added for 1 h. The cells were washed three times with PBS, and observed using a fluorescent confocal microscope (Zeiss, Germany) and were quantified by ImageJ software (Version 1.52, USA).

Western blotting analysis

Cells or animal tissues were lysed on ice with RIPA lysis buffer containing a protease phenylmethylsulfonyl fluoride and phosphatase inhibitor cocktail. Cytosolic and nuclear fractions were isolated using the Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, 78833) according to the manufacturer’s protocol.

Proteins were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% nonfat milk, the membrane was probed with the primary and secondary antibodies. The protein signals were visualized using an ECL kit and detected by an image reader (GE AI600, USA). All blots were processed in parallel and derive from the same experiment.

Statistics

All experimental data are presented as mean ± standard error of the mean (SEM). Differences among groups (≥3 groups) were analyzed using one-way ANOVA or differences between two groups using Student’s t test by GraphPad Prism 7 software (GraphPad Software, USA). Values of P < 0.05 were considered statistically significant.