HEK293T, SH-SY5Y, MEF AMPK WT, MEF AMPK DKO, HEK293 ATG5 WT, HEK293 ATG5 KO, Hela ATG8 WT, Hela ATG8 KO and PC12 cells were grown in DMEM medium (Hyclone, with L-glutamine, with 4.5 g/L glucose, without pyruvate); H4 cells were grown in DMEM medium (Hyclone, with L-glutamine, with 4.5 g/L glucose, with pyruvate). These media were supplemented with 10% FBS (GibcoTM), 1% Penicillin/Streptomycin (GibcoTM). Doxycycline-inducible 293THK stable cell lines were generated by co-transfecting HP138-HK2 and HP216 plasmids (a gift from Dr. Hui Yang) into HEK293T cells using Lipofectamine TM 2000 (InvitrogenTM) and selected with 10 µg/mL puromycin (Sangon® Biotech), obtained monoclonal cell lines by flow cytometry.
High throughput screening
FDA-approved drugs or drug candidates library were purchased from Topscience, Inc. (Shanghai, China). Drug concentration in the high-throughput screen was 10 μg/mL. Spautin-1 (10 μmol/L) and AC220 (2 μmol/L) treatment was used as a positive control to induce Chaperone mediated autophagy (CMA), DMSO was used as a negative control since compounds from the screen library were dissolved in DMSO. The inducers of CMA were screened using the 293THK stable cell line. The high-throughput screening was done using flow cytometry. Every plate has negative control (DMSO) and positive control (Spautin-1 + AC220), every plate has three replicates, and the screen Z-factor of every plate was > 0.5. There were two high-throughput screenings. In the first screening, 293THK cells were pretreated with or without DOX and were plated in 96-well plates at a density of 2 × 104 cells per well, treated with FDA-approved drugs or drug candidates for 24 h, the fluorescence of HK2-GFP was analyzed by flow cytometry. In the second screening, 293THK cells were pretreated with or without DOX and were plated in 96-well plates at a density of 2 × 104 cells per well, transfected with siRNA of Lamp2a for 48 h, then treated with the 195 hits from the primary high-throughput screening for another 12 h, the HK2-GFP fluorescence was analyzed by flow cytometry.
Reagents and antibody generation
The chemicals and their sources are as follows: Metformin (#A506198), DOX (#A603456), Puromycin (#A606719), NH4Cl (#A501569) from Sangon® Biotech; TPCA1 (#S2824), MG132 (#S2619), Bafilomycin A1 (#S1413), E-64D (#S7393), Leupeptin hemisulfate (#S7380), Spautin-1 (#S7888), AC220 (#S1526) from SelleckChem; Aβ25-35 (#A4559) from Sigma. Anti-Flag (DYKDDDDK) Affinity Gel (#B23102), Anti-HA magnetic beads (#B26202) and Anti-Myc magnetic beads (#B26301) were purchased from Bimake. Pierce™ Protein A/G Magnetic Agarose Beads (#78610) were purchased from Thermo Fisher Scientific. Lipofectamine TM 2000 (#1901433) and Lipofectamine TM 3000 (#2067450) were from Invitrogen. The following antibodies were used in this study: HK2 (#22029-1-AP), APP (#60342) from ProteinTech TM; GFP (B-2) (#sc-9996), Hsc70 (B-6) (#sc-7298) from Santa Cruz; β-tubulin (#M1305-2), Flag-Tag (#M1403-2), HA-Tag (#0906-1), Myc-Tag (#R1208-1) from HuaAn Biotechnology; Lamp2a (#ab18528) from Abcam; PKM2 (#4053), phospho-AMPKα (Thr172) (#2535), phospho-IKKα/β (ser176/180) (#2694), AMPKα (#5832), IKKα (#11930), IKKβ (#8943), APP/β-Amyloid (#2450), TAK1 (#5206), ATG5 (#12994S) from Cell Signaling Technology®. LC3 (#L8918) from Sigma. The anti-phospho-human Hsc70 ser85 antibody was raised against the region near Ser85 phosphorylation site of Hsc70. The phosphorylated synthetic peptide [DAVVQSDMKHWPFMC] was used for immunization in the rabbits. The antibody was generated by GenScript (Nanjing China). The secondary antibodies for Western blot were used: Goat anti-Mouse IgG (H + L) (#31430, Thermo Fisher Scientific), Goat anti-Rabbit IgG (H + L) (#31460, Thermo Fisher Scientific).
Molecular cloning and siRNA knockdown
Hsc70-Flag, Lamp2a-Flag, λ-phosphatase-Flag, IKKα-Myc, IKKβ-HA, APP, were cloned into pcDNA5 via BamHI and AvaI restriction enzymes by PCR amplifying the ORFs from cDNA templates of Hsc70 (#19514, Addgene), Lamp2a (#86146, Addgene), APP (#114193, Addgene), λ-phosphatase, IKKα and IKKβ were presented by Life Sciences Institute, Zhejiang University. Molecular cloning was performed using T4 DNA ligase and transformed into DH5α E. coli cells. Plasmid purifications and extractions were performed using the NucleoBond Xtra Midi kit (Macherey-Nagel). siRNA sequences were detailed in Table S1, and siRNA were transformed into HEK293T, H4, SH-SY5Y, 293THK, PC12 cells with Lipofectamine TM 3000 (Invitrogen), according to the manufacturer’s protocol.
The fluorescence of HK2-GFP was analyzed by flow cytometry. 293THK cells were pretreated with or without 1 μg/mL DOX and treated with indicated methods. The cells were analyzed by flow cytometry (Beckman Coulter Cytoflex), and data were analyzed by CytExpert2.3, the FlowJo software. 293THK without DOX was used as a negative control to distinguish between negative and positive fluorescence cells.
Protein purification and in vitro kinase assays
For purification of IKKα-Myc and IKKβ-HA, pcDNA5-IKKα-Myc and pcDNA5-IKKβ-HA plasmids were transfected into HEK293T cells for 24 h. Cells were lysed in 1 mL of lysis buffer (TAP) (20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.5% NP-40, 1 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L EDTA, Protease inhibitor cocktail (Bimake, added fresh)), and incubated with anti-Myc magnetic beads and anti-HA magnetic beads (after washing the beads with PBS, twice) for 6 h on a rotating wheel at 4 °C. The beads were washed three times with TAP buffer and treated with CIP (#M0290, Biolabs) for 30 min at 37 °C. The kinase assays were performed with recombinant Human Hsc70 proteins (#ab78431, Abcam). The proteins were incubated in 30 μL of kinase buffer (25 mmol/L Tris-HCl (pH 7.5), 5 mmol/L β-glycerophosphate, 2 mmol/L dithiothreitol (DTT), 0.1 mmol/L Na3VO4, 10 mmol/L MgCl2) supplemented with phosphatase inhibitor cocktail (#b15001, Bimake), with or without 100 μmol/L ATP for 30 min at 30 °C. The reactions were stopped by adding SDS-PAGE 2× loading buffer (100 mmol/L Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2 mol/L DTT, 0.1% bromophenol blue) and heating at 100 °C for 10 min.
Purification of TAK1
For purification of TAK1-Flag, pcDNA5-TAK1-Flag were transfected into HEK293T cells for 24 h. Cells were lysed in 1 mL of lysis buffer (TAP) (20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.5% NP-40, 1 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L EDTA, Protease inhibitor cocktail (Bimake, added fresh)), and incubated with anti-Flag magnetic beads (after washing the beads with PBS, twice) for 6 h on a rotating wheel at 4 °C. TAK1-Flag were eluted using 3× Flag peptide. The protein was dissolved into TBS and diluted to a final concentration of 50 μg/mL before the protein thermal shift assay.
Protein thermal shift assay
The protein thermal shift assay was performed as described previously (Groftehauge et al. 2015; Xu 2020). To determine stability, purified TAK1 were added to SYPRO Orange dye. Metformin and 5z-7-oxozeaenol were added to the proteins to make a final concentration of 20 mmol/L and 100 μmol/L and incubated at 4 °C for 1 h. The experiments were performed in 96-well plates specific for the real-time PCR instrument with a total volume of 20 μL/well. The assay plate was placed into the ABI-7500 Fast Real-time PCR system. The reaction was run from 25 °C, ramping up in increments of 0.05 °C/s to a final temperature of 99 °C with fluorescence detection throughout the experiment to generate a dataset. The melting temperatures of the protein was determined by performing nonlinear fitting of the data set to a Boltzmann sigmoidal curve in GraphPad Prism.
For purification of Hsc70-WT-Flag, Hsc70-S85A-Flag, Lamp2a-Flag, APP-WT, and APP-M7, pcDNA5-Hsc70-WT-Flag, pcDNA5-Hsc70-S85A-Flag, pcDNA5-Lamp2a-Flag, pcDNA5-APP, and pcDNA5-APP-M7 plasmids were transfected into HEK293T cells or SH-SY5Y cells for 24 h. Cells were washed with PBS and lysed in 1 mL of lysis buffer (TAP) (20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.5% NP-40, 1 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L EDTA, Protease inhibitor cocktail (Bimake, add fresh) for 30 min, incubated with anti-Flag (DYKDDDDK) beads or APP antibodies incubated with Pierce™ Protein A/G Magnetic Agarose Beads (#78610, Thermo Fisher Scientific) for 6 h on a rotating wheel at 4 °C. The beads were washed with TAP three times, 5 min each wash, and SDS-PAGE 2× loading buffer was added, followed by heating at 100 °C for 10 min.
Cell lysates or pull-down samples were added SDS-PAGE 2× loading buffer and heated at 100 °C for 10 min, subjected to 10%–12% SDS-PAGE, and then transferred onto PVDF membranes for 1 h at 0.2 A with semi-dry transfer system of BioRad. Membranes were blocked in PBST buffer containing 5% (w/v) skimmed milk for 1 h and probed with the indicated antibodies in PBST containing 5% (w/v) BSA at 4 °C overnight. Detection was performed using HRP-conjugated secondary antibodies and chemiluminescence reagents (#4AW001-500, 4A Biotech Co.). We used ImageJ 1.53a for the analysis of grayscale quantification. The numbers under the blots represent the average value (the ratio to Tubulin) of grayscale quantification.
Cell death and survival assays
SH-SY5Y cells were transfected with pcDNA5-APP or pcDNA5-APP-M7, co-transfected with or without pcDNA5-Hsc70-WT-Flag, or pcDNA5-Hsc70-S85A-Flag for 24 h using Lipofectamine TM 3000 (Invitrogen TM), treated with or without Metformin (20 μmol/L) for another 48 h. PC12 cells were transfected with siRNA of Hsc70, Lamp2a and ATG5, treated with or without 20 μmol/L Aβ25-35, and Metformin (20 μmol/L) for 96 h. PC12 cells were treated with Aβ25-35 (20 μmol/L) and Metformin (20 μmol/L) for 72 h, with or without TPCA1 (5 μmol/L) for another 24 h. Cell viability was detected using CellTiter-Glo® Luminescent Cell Viability Assay (#G7573 Promega) according to the manufacturer’s instructions.
Mass spectrometry and data analysis
Hsc70 was trypsin-digested on beads following immunoprecipitation. The resulting peptides were subjected to the phosphopeptide enrichment using TiO2 beads. The enriched phospho-peptides were analyzed on the Q Exactive™ HF mass spectrometer (Thermo Fisher Scientific). The identification and quantification of phosphorylated peptides were done using MaxQuant (Cox and Mann 2008). The tandem mass spectra were searched against UniProt human protein database together with a set of commonly observed contaminants. The precursor mass tolerance was set as 20 ppm, and the fragment mass tolerance was set as 0.1 Da. The cysteine carbamidomethylation was set as a static modification, and the methionine oxidation, as well as serine, threonine, and tyrosine phosphorylation, were set as variable modifications. The FDR at peptide spectrum match level was controlled below 1%.
Proximity ligation assays (PLA)
PLA assay were performed according to Duolink (#DUO92010, Sigma). H4 cells or HEK293T cells were cultured on glass slides, treated with Metformin or transfected with indicated plasmids for the indicated times, washed twice with PBS, fixed with 4% paraformaldehyde for 20 min. Following blocking with 5% FBS supplement with 0.1% Triton X-100 to increase the permeabilization for 1 h, the primary antibodies (Hsc70, PKM2, Lamp2a, Flag) were incubated with the slides overnight at 4 °C. After incubating with the secondary antibodies conjugated with the PLA probes, the signals were amplified through ligation and amplification. The fluorescence analysis was done using Cytation® 3.
Male transgenic APP/PS1 (C57BL/6) mice at the age of 14–16 weeks were purchased from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China) and housed in a pathogen-free environment of experimental animal center in Zhejiang University. All animal studies and experimental procedures were approved by the Animal Care and Use Committee of the animal facility at Zhejiang University. Mice were divided into three groups: normal C57 group (WT, n = 7), control APP/PS1 group (APP/PS1 + Veh, n = 7), and APP/PS1 treated with Metformin group (APP/PS1 + Met, n = 7). The mice in the Metformin group had access to tap water with Metformin freely for 12 weeks.
For Hsc70 overexpression mice, 16 weeks old mice were divided into three groups: APP/PS1 mice injected AAV control (mCherry) group (Ctrl, n = 6), APP/PS1 mice injected AAV-Hsc70- WT-Flag-P2A-mCherry group (Hsc70 WT, n = 6), APP/PS1 mice injected AAV-Hsc70-S85A-Flag-P2A-mCherry group (Hsc70 S85A, n = 6). The AAV encoded mRNA of Hsc70 WT-Flag-P2A-mCherry, Hsc70 S85A-Flag-P2A-mCherry, and mCherry under the control of CAG promoter and packaged into an adeno-associated virus (AAV8) by Vigene biosciences, China. The stereotactic injection was performed in anesthetized mice, and AAV encoding mCherry, Hsc70 WT-Flag-P2A-mCherry or Hsc70 S85A-Flag-P2A-mCherry were injected into the hippocampus (1.5 μL bilateral) of APP/PS1 mice (A/P − 2 mm, M/L − 1.5 mm, D/V − 1.5 mm), and flow rates are 0.3 μL/min, as described previously (Wegmann et al. 2017), under standard aseptic surgery conditions. After finishing the injection, the needle was left in place for 2 min to allow the diffusion of the injected AAV solution. Close the skin incision using glue and let the mouse recover from anesthesia. Injected mice were housed under the standard condition for 12 weeks.
Morris water maze
The water maze behavioral tests were performed as described previously (Vorhees and Williams 2006). The device is a circular pool (140 cm diameter) filled with water supplemented with titanium dioxide and maintained at 22 °C. A 12 cm diameter platform was placed 1 cm below the water surface in a fixed position. Mice were trained in four quadrants every day for five consecutive days. Each quadrant trial lasted 60 s or until the mouse found the platform. If the mouse did not find the platform within the prescribed time, the experimenter needed to guide the mouse to stand on the platform for 20 s. All parameters were recorded by a video tracking system.
The brain samples were collected from the mice, hemisected, and fixed in 4% paraformaldehyde for 24 h. The following procedures were done by HUABIO, China. Tissues were stained with polyclonal rabbit anti-GFAP antibody (#Z033401-2, DAKO, 1:100) to detect astrocytes, mouse anti-β-Amyloid, 1-16 antibody (clone 6E10, #803002, Biolegend®, 1:100) to detect Aβ plaques, followed by secondary antibody staining and imaging with Cytation® 3.
ELISA for Aβ1-42
Mouse brain tissues were prepared according to the brain tissue homogenate protocol described in the ELISA technical guide from Life technologies. The accumulation of Aβ1-42 was quantified by ELISA (# KHB3441, Thermo Fisher Scientific).
The prediction of Hsc70 kinases was made using GPS3.0 software (Xue 2008) using the “Medium” threshold setting. APP amino acid sequences were obtained from NCBI and aligned by Clustal W. The alignment images were obtained using the ESPript 3.0 software. ImageJ 1.53a was used for grayscale quantification of immunoblotting.
Quantification and statistical analysis
Statistical analyses were performed with GraphPad Prism 7. Data were analyzed with a one-way analysis of variance (ANOVA) test or Student’s t-test. Data points are shown as mean ± SD. All experiments contained at least three biological replicates.