The following reagents were purchased from the indicated providers: dimethyl sulfoxide (DMSO; Sigma, D8418), MG132 (Calbiochem/Merck-Millipore, 474791), rotenone (Sigma, R8875), rapamycin (InvivoGen, tlrl-rap), ULK1 inhibitor (MRT68921; Selleckchem, S7949), and mifepristone (RU-486; Sigma, M8046). We also received HEXA-018 from Hexa Pharmatec., which is not commercially available (Patent number, WO 2020/017878 A1).

The following antibodies were used for immunoblotting: GFP (Clontech, 632380), p62 (Sigma, P0067), LC3 (MBL, PM036), phospho-mTOR Ser2448 (Cell Signaling, 5536), phospho-mTOR Ser2481 (Cell Signaling, 2974), mTOR (Cell Signaling, 2983), phospho-ULK1 Ser757 (Cell Signaling, 6888), ULK1 (Abcam, ab128859), phospho-AMPKα1 Thr183 + AMPKα2 Thr172 (GeneTex, GTX130429), AMPKα1/α2 (GeneTex, GTX50863), Ref2(P) (Abcam, ab178440), HRP-conjugated anti-alpha-tubulin (Cell Signaling, 9099), HRP-conjugated rabbit IgG (Santa Cruz Biotechnology, sc-2004), and HRP-conjugated mouse IgG (Santa Cruz Biotechnology, sc-2005).

The Neuro-2a (ATCC, N2a) mouse neuroblastoma cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, 11995-065) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, 16000-044) and 50 μg/ml penicillin-streptomycin (Gibco, 15140-122). Cells were grown at 37°C in a humidified atmosphere containing 5% CO₂. All experiments were performed using cells from passages 5 to 15.

Primary cultures of cerebral cortical neurons were prepared from 16-days embryonic mice as described previously (Enokido et al., 1992; Araki et al., 2000). Briefly, mouse embryos were decapitated, and the brains were rapidly removed and placed in a culture dish containing HBSS (Gibco, 14170-112). Cortices were isolated, transferred to a conical tube and washed twice in HBSS. Cortical tissues were enzymatically digested by 20 units/ml papain (Worthington Biochemical Corporation, LK003150) and 0.005% DNase I for 30 min at 37°C. The tissues were mechanically dissociated by gently pipetting up and down. The cortical cells were centrifuged at 800 × g for 10 min at room temperature. Then, the dissociated cells were seeded on plates coated with poly-D-lysine (Sigma-Aldrich, P7405) in neurobasal media containing 2 mM glutamine (Gibco, 25030-081), N2 supplement (Gibco, 17502-048), B27 supplement (Gibco, 17504-044), and penicillin-streptomycin. The culture media were changed initially after 5 days and then every 3 days. Animals used in the current research were acquired and cared for in accordance with the guidelines published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

N2a cells (3 × 10⁴ cells/ml) or primary neurons (8 × 10⁴ cells/ml) were grown in 96-well plates and treated with MG132/rotenone or drugs as indicated for 24 h. DMSO was used as a negative control. For measurement of cytotoxicity, Cell Counting Kit-8 (CCK-8; Enzo Life Science, ALX-850-039-KI02) was used according to the manufacturer’s instructions. Briefly, 10 µL of CCK-8 reagent was added to each well, and the plate was incubated at 37°C for 2 h. The absorbance at 450 nm was measured by using a microplate reader (Tecan). Cell viability is expressed as a percentage of the control. All experiments were performed in triplicate.

N2a (30 × 10⁴ cells/ml) or primary neurons (80 × 10⁴ cells/ml) were detached with trypsin-EDTA and washed twice with cold PBS. The cells were then resuspended in 250 µL of binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂ (pH 7.4)) and incubated with 3 µL of FITC-conjugated Annexin V (apoptotic cells; BD Biosciences) according to the manufacturer’s guide. Then, cells were gently vortexed and incubated for 15 min at room temperature in the dark. After adding Propidium iodide (necrotic cells; 20 μg/ml), flow cytometry was performed within 1 h using MoFlo Astrios (Beckman Coulter).

N2a cells (20 × 10⁴ cells/ml) or primary neurons (40 × 10⁴ cells/ml) were treated with drug for 8 h, and RNA was extracted from cells by using TRIzol plus RNA Purification Kit (Invitrogen, 12183-555) according to the manufacturer’s instructions. cDNA synthesis was performed at 37°C for 120 min with 100 ng of RNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, 4368814). Quantitative RT-PCR was performed using the one-step SYBR^® PrimeScript™ RT-PCR kit (Takara Bio Inc, RR420A) according to the manufacturer’s instructions, followed by detection using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems). 18S rRNA was used as an internal control. The 2^−ΔΔCt method was used to calculate relative changes in gene expression, as determined by real time PCR experiments (Livak and Schmittgen, 2001).

Cells or 20 adult fly heads were homogenized in Cell Lysis Buffer (Cell Signaling Technology, 9803) with protease and phosphatase inhibitor cocktails. The protein concentration of the cell or fly head lysates was determined by a BCA protein assay (Thermo Fisher, 23225). Next, the protein extracts were mixed with 4x Bolt LDS Sample Buffer (Invitrogen) and 10x Bolt Sample Reducing Agent (Invitrogen) and then boiled at 95°C for 5 min. An equal amount of protein from each sample was separated on Bolt 4–12% Bis-Tris gels (Invitrogen, NW04120BOX) or NuPAGE 3–8% Tris-Acetate gels (Invitrogen, EA0378BOX) and transferred to a polyvinylidene difluoride membrane (PVDF; Invitrogen, LC2005). After the membrane was blocked with 5% skim milk in TBS with 0.025% Tween 20, it was probed with antibodies as indicated and detected with an ECL Prime Kit (GE Healthcare, RPN2232). Samples from three independent experiments were used, and the relative expression levels were determined using a Fusion-FX system (Viber Lourmat).

N2a cells (3 × 10⁴ cells/ml) or primary neurons (8 × 10⁴ cells/ml) were treated with rapamycin or drugs for 24 h, and the cells were assessed using the Cyto-I Green Autophagy Kit (Enzo Life Science, ENZ-51031-K200) according to the manufacturer’s instructions. Briefly, Cyto-ID dye or Hoechst 33342 was added to each well of a 96-well plate. Then, the plate was incubated at 37°C for 30 min. Cells were washed in 1x Assay Buffer with 2% FBS. The fluorescence signals (excitation/emission 480/530 nm and 340/480 nm) were measured by using a FlexStation 3 Microplate Reader (Molecular Devices). The ratios of the 480/530 signals over the 340/480 signals were calculated for each sample, and the Cyto-ID fluorescence is represented as a percentage of the control. All experiments were performed in triplicate.

Instead of pellet embedding, flat embedding was used for autophagy assessment in cells (Ylä-Anttila et al., 2009). Without cell harvest, cells on coverslips were fixed at 4°C for 1 h in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and postfixed with 2% reduced osmium tetroxide (3% potassium ferrocyanide combined with an equal volume of 4% osmium tetroxide) for 30 min at 4°C. Then, the cells were stained with thiocarbohydrazide (TCH) and 2% osmium tetroxide in distilled water and en bloc in 1% uranyl acetate. The cells were then dehydrated via a graded ethanol series and embedded with an EMBed-812 Embedding Kit (EMS). The embedded samples were incubated for 48 h in 60°C. Resin blocks were incubated for 48 h at 60°C. The embedded samples were sectioned (60 nm) with an ultramicrotome (Leica), and the sections were then viewed on a Tecnai 20 transmission electron microscope (TEM; Thermo Fisher) at 120 kV. Images were captured with a US1000X-P camera 200. Stitching images were acquired using Photomontage software (Thermo Fisher). The numbers of autophagosomes and autolysosomes were counted in cells of almost the same size using ImageJ software (National Institutes of Health).

For assessment of neuronal mitochondrial dysfunction, N2a cells (4 × 10⁴ cells/ml) and primary neurons (8 × 10⁴ cells/ml) were seeded in XF24-well culture plates (Seahorse Bioscience). The cells were washed twice with XF Base Medium supplemented with 2 mM L-glutamine, 10 mM D-glucose and 1 mM sodium pyruvate (pH 7.4) and incubated at 37°C in a non-CO₂ incubator for 1 h. Mitochondrial dysfunction was evaluated using the XF Cell Mito Stress Test Kit (Seahorse Bioscience) according to the manufacturer’s instructions, followed by measurement using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience). The 24-well utility plate was hydrated, treated with 2 µM oligomycin, 2 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and 0.5 µM antimycin A + rotenone, and was then used to calibrate the analyzer. The basal oxygen consumption rate (OCR), ATP production, maximum reserve, and respiratory capacity were calculated as previously described (Dranka et al., 2011), with averages calculated from 4 wells per condition in each individual experiment. The OCR was normalized to the total protein concentration (OD). After the Seahorse analysis, the plate was centrifuged at 280 × g for 5 min. The media were aspirated, and the plate was washed twice with PBS. The cells were lysed in RIPA buffer. Protein concentrations in cell lysates were determined using a BCA assay kit.

N2a cells (2.5 × 10³ cells/ml) were plated in 96-well plates and transfected with Gfp (pCMV-AC-Gfp) and human TDP-43 (pCMV-AC-TDP-43-Gfp) cDNA by using Lipofectamine 3000 reagent (Invitrogen, L3000-015) according to the manufacturer’s instructions. Six hours after transfection, the cells were treated with HEXA-018 (5 µM) and subsequently treated with IncuCyte Red Cytotoxicity Reagent (50 nM; EssenBioscience, 4632). Images were collected with an IncuCyte Zoom System and a ×20 objective lens at 6 h intervals. Cell toxicity was analyzed with IncuCyte ZOOM software by counting the green and red double-positive cells.

Drosophila stocks were maintained on standard cornmeal agar media at 24°C unless otherwise noted. UAS-TARDBP was described previously (Kim et al., 2014). All other stocks were from The Bloomington Stock Center.

Adult males (0–1 day old) were separated and transferred into experimental vials containing fly medium mixed with or without RU-486 at a density of 25 (for lifespan) or 25 (for climbing assay) flies per vial (n > 100). The number of dead flies was recorded daily, and the flies were transferred to fresh media every other day. Adult locomotor function was assessed by a previously described method (Feany and Bender, 2000), with 100 flies per genotype per time point in all experiments. Experiments were repeated twice to ensure consistent results.

To investigate whether HEXA-018 activates the ALP, we examined the microtubule-associated protein light chain 3 (LC3-I/II) ratios in N2a cells following treatment with HEXA-018. The increase in the LC3-I/II ratio is a typical indicator of autophagic activation. We found that HEXA-018 treatment significantly increased the LC3-I/II ratio in N2a cells (Figure 1B). Another features of ALP activation are the reduction of p62 protein. However, HEXA-018 did not affect the protein level of p62 in neuronal cells (Figure 1B). We also observed that HEXA-018 treatment upregulated the transcription of lc3a and lc3b mRNA in N2a cells and primary neurons (Figure 1C). We confirmed these results in primary neurons treated with HEXA-018 (Figures 2D,E). For further confirmation of ALP activation, we used Cyto-ID fluorescence dye. Cyto-ID fluorescence dye specifically labels all types of autophagic vacuoles, including amphisomes or autolysosomes (Guo et al., 2015). Thus, the fluorescence intensities of Cyto-ID-stained cells indicate the level of ALP activation. Rapamycin is a well-known activator of the ALP (Thellung et al., 2019), so the fluorescence intensity of Cyto-ID-stained N2a cells and mouse primary neurons was strongly elevated by rapamycin treatment (Figures 1F,G). Consistent with these results, we observed that the HEXA-018-treated cells showed increased Cyto-ID fluorescence intensities in both N2a cells and primary neurons (Figures 1F,G). These results suggest that HEXA-018 activates the ALP in N2a cells and primary neurons.

The most well-studied regulatory mechanism of the ALP is the mTOR (mechanistic target of rapamycin) pathway. To determine whether HEXA-018 induces autophagy via modulation of the mTOR pathway, we measured the phosphorylation level of mTOR in the HEXA-018-treated N2a cells and primary neurons. The phosphorylation of mTOR (Ser2481 and Ser2448) is critical for mTOR activation. However, the phosphorylation levels of mTOR were not affected by HEXA-018 treatment in N2a cells (Figures 2A,B). We confirmed these results in primary neurons treated with HEXA-018 (Figures 2C,D). These results suggest that HEXA-018 activates the ALP in an mTOR-independent manner. Previous studies demonstrated that the phosphorylation of ULK1 (Ser757) and AMPK1 (Thr183 and Thr172) is the major contributor of ALP activation in mTOR dependent and independent ALP (Jung et al., 2010; Roach, 2011; Din et al., 2012). We analyzed phosphorylation levels of ULK1 and AMPK1 in N2a cells and primary neurons treated with HEXA-018. We showed that HEXA-018 clearly increased the phosphorylation levels of ULK1 and AMPK1 (Figures 2A–D). We used rapamycin as a mTOR-dependent activator of autophagy. We found that the rapamycin treatment significantly decreased the mTOR phosphorylation, but HEXA-018 treatment did not change mTOR phosphorylation in rapamycin-treated neuronal cells (Figures 2E,F). Consistent with previous findings, we observed that rapamycin decreased the level of p62 protein in N2a cells and primary neurons (Supplementary Figures 1A,B). These results suggest that HEXA-018 significantly increased the ULK1 and AMPK1 phosphorylation levels, but mTOR phosphorylation levels were not affected by HEXA-018 treatment. In addition, HEXA-018 induced autophagy activation was significantly decreased by ULK1 inhibition in N2a cells and primary neurons (Figures 2G,H). Taken together, HEXA-018 activates the ALP via the ULK1-AMPK pathway in an mTOR-independent manner.

Electron microscopy is one of the most precise ways to detect and quantify autophagic structures. To investigate whether HEXA-018 increases the number of autophagic vacuoles, we analyzed autophagic structures using transmission electron microscopy (TEM). With autophagy initiation, the phagophore fully surrounds its cargo and fuses to form the double-membrane autophagosome. After fusion with the lysosome, strong electron density represented as the criterion for identification of autolysosomes due to degradation of materials. Various autophagic structures, including autophagosomes and autolysosomes, were observed in N2a cells. The red arrows in Figure 3A indicate autophagic structures. The number of autophagic vacuoles was substantially increased after HEXA-018 or rapamycin treatment in N2a cells (Figure 3A). To investigate the regulatory role of HEXA-018 in autolysosome formation, we fluorescent puncta assay with DsRed-LC3-GFP, which is a well-established method to monitor autophagy flux (Sheen et al., 2011). This assay uses the characteristics that the GFP fluorescence is lost but DsRed fluorescence is stable in the lysosomal acidic environment. The autophagic vesicles, both autophagosome (yellow puncta) and autolysosome (red puncta), were significantly increased by HEXA-018 and rapamycin treatment in N2a cells (Supplementary Figure S2). We then further assessed the numbers of autophagosomes and autolysosomes in the control and HEXA-018-treated cells using TEM (Figure 3B). The number of autophagosomes was not significantly changed, but the number of autolysosomes was significantly increased in the HEXA-018-treated cells compared with the control cells (Figure 3B). These results strongly suggest that HEXA-018 activates the ALP. Considering that autolysosome formation was significantly increased by HEXA-018 treatment, HEXA-018 appears to promote the fusion of autophagosomes and lysosomes.

MG132 (proteasome inhibitor) and rotenone (inducer of reactive oxygen species (ROS)) are currently accepted as neurotoxicity-inducing factors. Previous studies have shown that ALP activation mitigates MG132-or rotenone-induced neurotoxicity. Thus, we investigated the protective effect of HEXA-018 in MG132-or rotenone-treated N2a cells and primary neurons. HEXA-018 significantly attenuated the cytotoxicity of MG132 and rotenone in N2a cells (Figures 4A,B; Supplementary Figures 3A,B) and primary neurons (Figures 4C,D; Supplementary Figures 3C,D). We also confirmed these results using a different experimental approach based on flow cytometry using Annexin V and PI staining. We found that HEXA-018 attenuated the rotenone/MG132-induced necrotic cell death in N2a (Figure 4E; Supplementary Figure 4A) and primary neurons (Figure 4F; Supplementary Figure 4B). These results indicate that HEXA-018 mitigates UPS impairment and ROS-induced neurotoxicity.

Recent studies have suggested that overproduction of ROS leads to mitochondrial damage and that mitochondrial dysfunction is a key pathological feature of many neurodegenerative diseases, such as ALS, AD, and PD. Accumulating evidence suggests that autophagy activation suppresses rotenone-induced neurotoxicity such as mitochondrial dysfunction and oxidative stress in cell and mice (Chen et al., 2007; Lin et al., 2012; Xiong et al., 2013). Moreover, rotenone-induced α-synuclein aggregates are significantly decreased by rapamycin treatment (Yu et al., 2009). Thus, we next investigated whether HEXA-018 suppresses rotenone-induced mitochondrial dysfunction in neuronal cells. N2a cells and primary neurons were treated with HEXA-018 and rotenone, and then, we measured the cellular oxygen consumption rate (OCR) using the Seahorse XF24 Extracellular Flux Analyzer and a mitochondrial stress test kit. OCR is an indicator of mitochondrial respiration, including basal respiration, ATP production, maximal respiration, and spare respiratory capacity. Analysis of mitochondrial respiratory parameters was performed by using oligomycin, FCCP, and antimycin A + rotenone. Notably, we found that the basal respiration, ATP production, and maximal respiratory parameters were markedly decreased in the rotenone-treated cells compared to the control cells. The rotenone-induced reductions in basal respiration, ATP production, and maximal respiration parameters were strongly ameliorated by HEXA-018 treatment, but the spare respiratory capacity was not altered (Figures 5A–D). Taken together, these findings suggest that HEXA-018 attenuates rotenone-induced mitochondrial dysfunction in neuronal cells.

Previous studies have indicated that overexpression of TDP-43 cause neuronal toxicity in mammalian and Drosophila neurons. In addition, oxidative stress and UPS impairment are key pathological features in TDP-43 proteinopathy. All these data suggest that HEXA-018 might attenuate TDP-43-induced neurotoxicity. Thus, we analyzed TDP-43 toxicity using a live cell imaging system (IncuCyte). GFP- or GFP-tagged TDP-43-expressing N2a cells were treated with HEXA-018, and the cell death of the GFP-expressing cells was monitored. As expected, TDP-43-induced neuronal toxicity was significantly suppressed by HEXA-018 treatment (Figure 6A).

We also assessed ALP activity in a Drosophila in vivo model. To evaluate ALP function, we expressed the Atg8a-GFP reporter in the fly nervous system. The ratio of free GFP to Atg8a-GFP correlates with ALP activity because globular GFP protein is much more resistant to acidic lysosomal conditions than Atg8a. By adopting this method, we examined the free GFP/Atg8a-GFP ratio in the fly model. We found that the ratio was significantly higher in the HEXA-018-treated flies than in the control flies (Figure 6B). Moreover, we found that Ref2(P) (Drosophila homologue of p62) protein level was not changed in Atg8a-GFP flies (Figure 6C). These data suggest that HEXA-018 activates the ALP in fly neurons. Given the strong in vitro evidence that HEXA-018 attenuates TDP-43-induced toxicity in neuronal cells, we next examined whether HEXA-018 treatment suppresses TDP-43-induced toxicity in a Drosophila model of TDP-43 proteinopathy that expresses human TDP-43 in the nervous system (Kim et al., 2014; Lee et al., 2020a). We also found that HEXA-018 treatment significantly improved the TDP-43-induced climbing deficit and shortened lifespan compared with the control treatment (Figures 6D,E). Taken together, these results show that HEXA-018 ameliorates TDP-43 toxicity in fly and mammalian cell line models of TDP-43 proteinopathy via ALP activation.