Peripheral blood mononuclear cells (PBMCs) were freshly isolated from the whole blood of anonymous healthy human blood donors using Lymphoprep (Cat. No. 07851, Stemcell Technologies). Usage of healthy human blood received approval from the HKBU Research Ethics Committee (#REC/19-20/0110). CD14⁺ monocytes were isolated from the freshly prepared PBMCs using CD14 microbeads, human (Cat. No. 130-050-201, Miltenyi Biotec) according to manufacturer protocol. To generate MoDCs, CD14⁺ cells were cultured at a density of 1 × 10⁶ cells/ml in RPMI 1640 medium (Cat. No. 11875-085, Gibco) supplemented with 10% Fetal Bovine Serum (FBS) (Cat. No. 10270-106, Gibco), 1% Penicillin–Streptomycin (10,000 U/ml) (Cat. No. 15140-122, Gibco), 25 µg/ml rhIL-4 (Cat. No. 200-04, PeproTech), and 25 µg/ml rhGM-CSF (Cat. No. 300-03, PeproTech) for 5–7 days, with 50% medium change every 3 days. Cells were incubated at 37°C, 5% CO₂.

MoDCs were seeded at a density of 1 × 10⁶ cells/ml in RPMI 1640 medium 1% FBS in 24-well cell culture plate overnight at 37°C, 5% CO₂. C1 (1 µM) and Celastrol (0.25 µM) were added to pre-treat MoDCs for 1 h and recombinant Human Alpha-synuclein protein aggregate (Active) (fibrillar α-syn) (Cat. No. ab218819, Abcam) (1 µg/ml) was used to treat MoDCs for another 4 h. Cells were harvested, and total RNA was extracted using RNAiso Plus followed by cDNA generation by PrimeScript™ RT Master Mix (Cat. No. RR047A, Takara). Quantitative real-time PCR was carried out with the TB Green Premix Ex Taq II (Tli RNase H Plus) (Cat. No. RR820A, Takara) using StepOnePlus™ Real Time System (Cat. No. 4376600, Invitrogen). The following primer pairs were used: GAPDH, 5′-ACAGTCCATGCCATCACTGCC-3′, 5′-GCCTGCTTCACCACCTTCTTG-3′; IL-1β, 5′-ATGATGGCTTATTACAGTGGCAA-3′, 5′-GTCGGAGATTCGTAGCTGGA-3′; IL-6, 5′-AGACAGCCACTCACCTCTTC-3′, 5′-AGTGCCTCTTTGCTGCTTTC-3′; IL-23, 5′-TTTTCACAGGGGAGCCTTCT-3′, 5′-ACTGAGGCTTGGAATCTGCT-3′; TNF-α, 5′-GTCAACCTCCTCTCTGCCAT-3′, 5′-CCAAAGTAGACCTGCCCAGA-3′; TGF-β, 5′-CACGTGGAGCTGTACCAGAA-3′, 5′-GAACCCGTTGATGTCCACTT-3′; IL-10, 5′-GACTTTAAGGGTTACCTGGGTTG-3′. 5′-TCACATGCGCCTTGATGTCTG-3′. Relative expression was calculated by normalizing to GAPDH and by ΔΔCT method. The ΔΔCT method was used to calculate the relative expression of each gene with reference to GAPDH, which were then normalized to α-syn only treatment.

Mouse BV2 microglia cell line was purchased from Elabscience (Cat. No. EP-CL-0493) and cultured in DMEM, high glucose (Cat. No. 11965-126, Gibco) supplemented with 10% FBS. Cells were incubated at 37 °C, 5% CO₂ and subcultured when cell confluency reached 80–90%. Before treatment, BV2 were seeded in DMEM medium with 10% FBS in 24-well cell culture plate overnight at 37 °C, 5% CO₂. The next day, lipopolysaccharide (LPS) (100 ng/ml) was used to prime the cells for 3 h in DMEM medium with 10% FBS and washed 3 times with PBS and replaced with fresh DMEM medium. Then, fibrillar α-syn in a concentration of 1 µg/ml was used to treat the BV2 cells for 3 h. Cells were harvested and subjected to qRT-PCR using similar methods as above.

MoDCs were seeded at a density of 1 × 10⁵ cells/ml in RPMI 1640 medium 1% FBS in 24-well cell culture plate overnight at 37°C, 5% CO₂. C1 (1 µM) and Celastrol (0.25 µM) were added to pre-treat MoDCs for 1 h followed by the addition of fibrillar α-syn (1 µg/ml) for 24 h. In some cases, TNF-α (50 ng/ml) was added together with fibrillar α-syn. LPS (100 ng/ml) was used as the positive control. Afterward, MoDCs were collected and washed with fluorescence-activated cell sorter (FACS) buffer (PBS + 1% FBS) and incubated for 30 min at 4°C in 100 µl FACS Buffer with BV421 Mouse anti-Human HLA-ABC (Cat. No. 565332, BD Pharmingen), APC Mouse anti-Human HLA-DR (Cat. No. 560896, BD Pharmingen), PE-Cy7 Mouse anti-Human CD86 (Cat. No. 561128, BD Pharmingen) and FITC Mouse anti-Human CD80 (Cat. No. 555683, BD Pharmingen) antibodies. Flow cytometry was performed following standard protocols on a BD FACSCanto™ II cytometer.

α-Syn peptide-specific CD4⁺ T cells were generated according to the previous protocol (50) with some modifications. Briefly, freshly isolated PBMCs (5 × 10⁶ cells/ml) were first cultured in RPMI + 10% FBS + 1% GlutaMAX (Cat. No. 35050-061, Gibco) and stimulated with α-syn peptide EQVTNVGGAVVTGVT (5 µg/ml) (ChinaPeptides) with reference to previous studies (22). One day after stimulation, rhIL-2 (100 IU/ml) (Cat. No. 200-02, Preprotech) was added to the cell culture for 7 days, with 50% media change and replenishment of rhIL-2 every 3 days. Frozen autologous PBMCs were stimulated with the same α-syn peptide (5 µg/ml) overnight and were added into the original PBMCs culture in a ratio of 1:10 for re-stimulation for another 7 days, with 50% media change and replenishment of rhIL-2 every 3 days. Simultaneously, MoDCs were generated as mentioned above and cultured for 6 days. Prior to the co-culture experiment, MoDCs were pre-treated with either C1 (1 µM) or Celastrol (0.25 µM) for 1 h followed by treating with fibrillar α-syn (1 µg/ml) overnight in RPMI + 10% FBS + 1% GlutaMAX. Also, rhIL-2 concentration of the PBMCs culture was reduced to 20 IU/ml to minimize its effect on T cells but to maintain survival. On the day of the co-culturing experiment, total CD4⁺ T cells were isolated from the PBMCs culture using CD4⁺ T Cell Isolation Kit, human (Cat. No. 130-096-533, Miltenyi Biotec) and added into the MoDCs culture in a ratio of MoDCs:T cell = 1:5 and cultured for 1 day or 3 days. α-Syn-specific CD4⁺ T cell only was served as the negative control. One day after the co-culture experiment, half of the suspension cells were harvested for flow cytometry analysis of surface CD3, CD4, CD25, and intracellular IFN-γ and T-bet expression using FITC Mouse anti-Human CD3 (Cat. No. 555339, BD Pharmingen), V500 Mouse anti-Human CD4 (Cat. No. 560768, BD Pharmingen), APC-Cy7 Mouse anti-Human CD25 (Cat. No. 557753, BD Pharmingen), PE-Cy7 Mouse anti-Human IFN-γ (Cat. No. 557643, BD Pharmingen) and PerCP-Cy5.5 Mouse anti-Human T-bet (Cat. No. 561316, BD Pharmingen) antibodies. While on day 3 of the co-culturing experiment, the remaining suspension cells were harvested for flow cytometry analysis of surface CD3, CD4, CD25, and intracellular IL-17A, RORγt and FoxP3 expression using FITC Mouse anti-Human CD3 (Cat. No. 555339, BD Pharmingen), V500 Mouse anti-Human CD4 (Cat. No. 560768, BD Pharmingen), APC-Cy7 Mouse anti-Human CD25 (Cat. No. 557753, BD Pharmingen), Alexa Fluor 647 Mouse anti-Human RORγt (Cat. No. 563620, BD Pharmingen), PerCp-Cy5.5 Mouse anti-Human IL-17A (Cat. No. 560799, BD Pharmingen) and PE Mouse anti-Human FoxP3 (Cat. No. 560046, BD Pharmingen) antibodies. Flow cytometry was performed following standard protocols on a BD FACSCanto™ II cytometer.

MoDCs (1 × 10⁵ cells/200 µl) were seeded on Nunc™ Lab-Tek™ II 8-well Chambered Coverglass w/non-removable wells (1.5 Borosilicate Glass) (Cat. No. 155409, Invitrogen) in RPMI with 1% FBS overnight at 37 °C, 5% CO₂. For studying endo-lysosomal pathway, C1 (1 µM) and Celastrol (0.25 µM) were added to pre-treat MoDCs for 1 h followed by the addition of 1 µg/ml fibrillar α-syn for 15, 30, and 60 min. Cells were then fixed with 2% paraformaldehyde (PFA) for 20 min at room temperature, permeabilized using the blocking and permeabilizing buffer composed of PBS with 5% Normal Goat Serum (Cat. No. 31873, Invitrogen), 3% Bovine Serum Albumin (BSA) (Cat. No. 9048-46-8, Sigma) and 0.5% Triton X-100 for 20 min, 4 °C. Following washing, primary antibodies were used for overnight incubation in the staining buffer of PBS with 3% BSA and 0.1% Triton X-100 overnight at 4°C: for early endosome markers, Mouse anti-Rab5 (1:200) (Cat. No. ab66746, Abcam) and Rabbit anti-Rab14 (1:200) (Cat. No. ab40938, Abcam) were incubated with 15 min samples; late endosome marker, Mouse anti-Rab7 (1:500) (Cat. No. ab50533, Abcam) and recycling endosome maker Rabbit anti-Rab11 (1:500) (Cat. No. ab36112, Abcam) were incubated with 30 min samples; lysosome markers, Mouse anti-Rab9 (1:500) (Cat. No. MA3-067, Invitrogen) and Rabbit anti-Lamp1 (1:300) (Cat. No. ab24170, Abcam) were incubated with 60 min samples. All samples were also probed with Chicken anti-α-syn (1:1,000) (Cat. No. ARG10689, Arigobio) in the staining buffer of PBS with 3% BSA and 0.1% Triton X-100 overnight at 4 °C.

For studying autophagic pathways, after C1 or Celastrol pre-treatment, MoDCs were treated with 1 µg/ml fibrillar α-syn for 4, 6 or 16 h. Cells were then fixed and permeabilized as mentioned above. Primary antibodies of early autophagosome marker, Rabbit anti-Beclin1 (1:200) (Cat. No. ab62557, Abcam) and early endosome marker, Mouse anti-Rab5 (1:500) were incubated with 4 h samples; autophagosome cargo protein p62 (SQSTM1), Mouse anti-SQSTM1/p62 (1:200) (Cat. No. ab56416, Abcam) was incubated with 6 h samples; autophagosome marker, Rabbit anti-LC3B (1:300) (Cat. No. NB100-2220, Novus Biologicals) and late endosome marker, Mouse anti-Rab7 (1:500) were incubated with 16 h samples. All samples were also probed with Chicken anti-α-syn (1:1,000) in the staining buffer of PBS with 3% BSA and 0.1% Triton X-100 overnight at 4°C.

Following primary antibody staining, cells were washed 3 times with PBS + 0.01% Triton X-100. Secondary antibodies Alexa Fluor 488 Goat anti-Rabbit (1:1,000) (Cat. No. A27034, Invitrogen), Alexa Fluor 647 Goat anti-Mouse (1:1,000) (Cat. No. A28181, Invitrogen) and DyLight anti-Chicken IgY H&L (1:500) (Cat. No. ab96948, Abcam) were used for fluorescence staining at room temperature for 1 h in darkness. Cells were washed 3 times again with PBS with 0.01% Triton X-100. Cell nuclei were stained with Hoechst 33258 (1:800) (Cat. No. ab228550, Abcam) for 20 min at 4 °C in darkness. Lastly, cells were mounted using ibidi mounting medium (Cat. No. 50001, ibidi) and signals were acquired using Leica TCS SP5 confocal microscopy and images were analyzed using the LAS AF software (Leica). The number of Rab5, Rab7, Rab9, Rab11, Rab14, Beclin1, LC3 positive puncta and their colocalizations with α-syn were counted and colocalization intensity values were analyzed using ImageJ software (http://imagej.nih.gov/ij/) (51).

Cells were collected and washed with PBS, followed by lysing with lysis buffer (10 mM Tris–HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1.25 µg/ml pepstatin A, 1 mM PMSF) on ice for 45 min. Lysates were then centrifuged at 13,000×g, 4°C for 30–45 min. Supernatants were collected and protein concentration was measured using Pierce™ BCA Protein Assay Kit (Cat. No. 23225, Thermo Scientific) according to the manufacturer’s instructions. Protein samples were mixed with 5× loading buffer (250 mM Tris–HCl (pH 6.8), 10% SDS, 30% glycerol, 5% β-mercaptoethanol, 0.02% bromophenol blue) and were boiled at 98°C for 5 min before being loaded into 10–12% SDS-PAGE gel for electrophoresis and transferred to Immobilon^®-P polyvinylidene difluoride (PVDF) (Cat. No. P2938, Sigma) membrane using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Membranes were blocked with 0.5% BSA and 5% blotting-grade blocker (Biorad) in PBS-T or TBS-T for 1 h at room temperature and blotted with aforementioned primary antibodies Rabbit anti-Rab5, Mouse anti-Rab7, Rabbit anti-Beclin1, Mouse anti-SQSTM1/p62, Rabbit anti-LC3B and anti-beta-Actin, clone RM112 monoclonal antibody (Cat. No. MABT523, Merck Millipore) (1:500–1:1,000) overnight at 4°C, followed by incubating with secondary antibodies HRP conjugate Donkey Anti-Rabbit antibody (Cat. No. AP182P, Merck Millipore) or HRP conjugated Goat Anti-Mouse antibody (Cat. No. AP130P, Merck Millipore) (1:10,000) for 1 h at room temperature. Membrane blots were developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Cat. No. 34580, Thermo Scientific) or SuperSignal™ West Femto Maximum Sensitivity Substrate (Cat. No. 34094, Thermo Scientific). Signals were detected by ChemiDoc (Bio-Rad) and band intensities were quantified using ImageJ software.

Immunoprecipitation of Rab5-GTP or Rab7-GTP from MoDCs using (Cat. No. 83701, NewEast Biosciences) and Anti-Active Rab7 Mouse Monoclonal Antibody (Cat. No. 26923, NewEast Biosciences) according to the manufacturer protocol with some modifications. Briefly, cells were lysed using 1× Assay/Lysis Buffer with 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1.25 µg/ml pepstatin A, 1 mM PMSF for 30–45 min on ice. Lysates were centrifuged at 13,000×g, 4°C for 30–45 min. Supernatants were collected and protein concentration was measured using Pierce™ BCA Protein Assay Kit according to manufacturer’s instructions. Afterward, lysates were incubated with either Mouse anti-Rab5-GTP or Mouse anti-Rab7-GTP primary antibody (1 µg) pre-conjugated with protein A/G agarose slurry (20 µl) in 500 µl of 1× Assay/Lysis Buffer with protease inhibitors for a maximum of 2 h, 4°C, with gentle rotation for immunoprecipitation. Afterward, 5× loading buffer was used to elute the targeted proteins by boiling at 98°C for 5 min and the protein lysates were subjected to SDS-PAGE as described above. Membranes were blocked with TBS-T + 3% BSA and incubated with either Rabbit anti-Rab5 or Mouse anti-Rab7 primary antibody (1:1,000) in TBS-T + 3% BSA overnight at 4°C, followed by incubating with Recombinant Protein G (HRP) Cat. No. (ab7460, Abcam) (1:1,000) in TBS-T + 3% BSA for 1 h at room temperature. Membrane blots were developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate or SuperSignal™ West Femto Maximum Sensitivity Substrate. Signals were detected by ChemiDoc (Bio-Rad) and quantified using ImageJ software. A total of 25 µg of total cell lysates were used as input control.

The putative protein targets associated with C1 and Celastrol were predicted using SwissTargetPrediction tool (http://www.swisstargetprediction.ch). The protein–protein interaction networks of the predicted targets were generated using the STRING database (http://www.string-db.org/) (52). Potential proteins and pathways that are related to antigen processing and presentation were identified and grouped. Details of the analysis can be found in Supplementary Figures 10–12 and Tables S1, S2 .

Data generated were from at least three independent experiments. Results were presented as the mean ± standard deviation (SD), unless specified. Statistical significance was calculated by the One-way ANOVA and Tukey’s multiple comparisons test were used to analyze differences among treatment groups, unless specified. A probability value of P <0.05 was considered statistically significant.

To understand the role of DC in stimulating α-syn-specific T cell responses, we first treated MoDCs with fibrillar α-syn, and evaluated the effect of C1 and Celastrol by pre-treatment. The expression of antigen presenting MHC-I (or HLA-A,B,C) and MHC-II (or HLA-DR), co-stimulatory CD80 and CD86, and also the inflammatory cytokine gene expression were assessed. MoDCs were pre-treated with C1 or Celastrol for 1 h before being treated with fibrillar α-syn for another 4 h. Gene expression of pro-inflammatory cytokines IL-1β, IL-6, IL-23, TNF-α, and anti-inflammatory IL-10 and TGF-β were assessed by qRT-PCR. When compared to the untreated cells, α-syn had no significant induction of cytokine gene expression ( Figure 1 ). However, treatment using the same α-syn on mouse microglia BV2 cells resulted in the upregulation of these cytokines ( Supplementary Figure 1 ). Interestingly, a significant reduction of IL-1β, IL-6, and TNF-α gene expression and a slight upregulation of anti-inflammatory IL-10 gene expression were observed with Celastrol pre-treatment while C1 had no effects ( Figures 1A, B, D, E ). Our results suggested that α-syn had minimal effects on inflammatory cytokine gene expression in MoDCs, but this can still be modulated by Celastrol.

We next determined the surface expression of MHC-I, MHC-II, CD80, and CD86 on fibrillar α-syn-treated MoDCs. After 24 h of treatment, we found that fibrillar α-syn had no significant effect on the expression of these molecules in both cell frequencies (%) or mean fluorescence intensities (MFI) when compared to the negative control ( Figure 2 and Supplementary Figure 2 ). TNF-α treated DCs can complement antigen uptake and DC maturation (53–55). In our experiments, however, though TNF-α led to upregulation of MHC-I, MHC-II, and CD86 in all treatments, the effect of subsequent α-syn stimulation appears to be negligible ( Supplementary Figure 3 ). C1 and Celastrol had no effects in this experiment. Therefore, α-syn did not result in MoDCs upregulation of antigen presentation molecules, co-stimulatory molecules, or cytokines.

Even though α-syn-treated MoDCs had minimal responses, we next sought to examine whether these cells are capable to induce T cell responses using a co-culture model. α-syn-specific CD4⁺ T cells were generated using a similar protocol (50, 56), then co-cultured (in a ratio of 5:1) with autologous α-syn-pulsed MoDCs with or without C1 or Celastrol pre-treatment. Flow cytometric analysis was performed to measure the abundance of T-bet⁺IFN-γ⁺ (Th1) on day 1, and RORγt⁺IL-17A⁺ (Th17) and CD25⁺FoxP3⁺ (Treg) cells on day 3, among CD3⁺CD4⁺ T cells after co-culture ( Supplementary Figure 4 ). Data showed ~30% Th1 cells, ~40% Th17 cells, and ~20% Treg cells were stimulated by α-syn-pulsed MoDCs ( Figures 3A–D ). Of interest, within the Treg population stimulated by α-syn-pulsed MoDCs, ~20% of the cells co-expressed RORγt ( Figure 3F ). Celastrol pre-treatment of α-syn-pulsed MoDCs significantly reduced the frequencies of these T cell subsets, with 4-fold and 3.5-fold reduction in Th1 and Th17 cell frequencies, respectively ( Figures 3B, C ). It also improved the Th17/Treg ratio as there was a 2-fold decrease in Treg abundance ( Figures 3D, E ). In contrast, C1 pre-treatment resulted in a modest decrease in Th17 cells. We further found that Celastrol, but not C1, resulted in decreased FoxP3⁻RORγt⁺ and FoxP3⁺RORγt⁺ subpopulations while increased FoxP3⁺RORγt⁻ cells among CD3⁺CD4⁺CD25⁺ cells ( Supplementary Figure 4B ). Therefore, although α-syn treatment of MoDCs did not trigger the upregulation of antigen presentation molecules, α-syn-specific Th1, Th17, and Treg cells were still induced. Moreover, Celastrol appears to counteract this likely through a mechanism that does not involve the classic surface expressed MHC-II and co-stimulatory molecules.

To understand how α-syn is trafficked in MoDCs leading to CD4⁺ T cell activation, and how Celastrol inhibited this, we next sought to examine the association of endo-lysosomal pathway proteins with α-syn in MoDCs. To this end, we tracked the colocalization of fibrillar α-syn with intracellular Rab5, Rab7, Rab9, Rab11, Rab14 proteins and lysosome marker Lamp1 at various timepoints using confocal microscopy. At 15 min, α-syn was colocalized with early endosomal marker Rab5, which implies that α-syn was likely uptaken through endocytosis by MoDCs ( Figure 4A ). At 30 min, trafficking to late endosomes occurred as α-syn was shown to colocalize with Rab7⁺ vesicles ( Figure 4C ). Another 30 min later, it appeared that α-syn containing vesicles were fused with lysosomes as they were colocalized with Lamp1 or Rab9, where the latter may mediate late endosome and lysosome fusion ( Figures 4E, G ). Interestingly, Celastrol increased the colocalization of α-syn with Rab5⁺ and Rab7⁺ vesicles ( Figures 4B, D ), but not with Rab9⁺ and Lamp1⁺ vesicles ( Figures 4F, H ). Besides, we also observed increased colocalization of α-syn with Rab14⁺ and Rab11⁺ vesicles under Celastrol treatment ( Supplementary Figure 5 ), which are mainly found on early endosomes and recycling endosomes, respectively. However, this was unlikely a consequence of increased endosome formation as Celastrol had no significant upregulation on the number of Rab5⁺, Rab7⁺, Rab9⁺, Rab14⁺, Rab11⁺ and Lamp1⁺ vesicles in MoDCs ( Supplementary Figure 6 ). On the other hand, C1 had no significant effect except decreased the number of Rab9 and α-syn colocalization ( Figure 4F ). These results suggest that α-syn could enter the endo-lysosomal pathway that may then be processed and generate α-syn peptides for antigen presentation.

The endo-lysosomal pathway can converge with the autophagic pathway through fusion of autophagosomes with late endosomes to form amphisomes, which the engulfed proteins can be directed for degradation when fused with lysosomes (57, 58). Rab5 can be involved in autophagosome formation and, conversely, Beclin1 is also associated with Rab5-mediated endosomal trafficking (59, 60). Thus, we next tested whether fibrillar α-syn is also trafficked to autophagic and amphisomal components in MoDCs. MoDCs were treated with fibrillar α-syn for 4 h or 16 h and assessed for colocalization of autophagy-related Beclin1 and LC3 with α-syn, and their association with Rab5 and Rab7 proteins, respectively. Expectedly, Beclin1 or LC3 were found colocalized with α-syn ( Figures 5A, C, E, G ). Rab5 or Rab7 were also found colocalized in these puncta, indicating the formation of amphisomes ( Figures 5A, D, E, H and Supplementary Figure 7 ). These data suggest that apart from the endo-lysosomal pathway, autophagic pathway is also associated with the trafficking of α-syn in MoDCs, where the formation of LC3⁺Rab7⁺ amphisomes occurred, indicating the convergence of the two pathways. Moreover, Celastrol, but not C1, markedly increased the number of Rab5/Beclin1/α-syn and Rab7/LC3/α-syn puncta ( Figures 5D, H ), and also the autophagic Beclin1⁺ and LC3⁺ puncta ( Supplementary Figures 8B, E ). Also, Rab7/LC3 amphisomes were increased by 2.2-fold under Celastrol treatment, suggesting that Celastrol can induce both the autophagic and amphisomal vesicles interaction with α-syn ( Supplementary Figure 8F ). To confirm the confocal microscopy data, western blot analysis was performed on MoDCs at 4 and 16 h post α-syn treatment. Beclin1 protein level was found to be downregulated following α-syn treatment compared to control but appears to be rescued by Celastrol treatment ( Figures 6A, B ). Similarly, while the protein level of LC3-I or LC3-II had no significant change after α-syn treatment, Celastrol increased the LC3-II : LC3-I conversion ratio ( Figures 6E, F ). Furthermore, we tested the expression of p62 in α-syn treated MoDCs, where its decreased expression indicates autophagy flux. We found that α-syn treatment upregulated p62 expression which was counteracted by Celastrol ( Supplementary Figures 9C, D ). Moreover, an increased p62 colocalization with α-syn in Celastrol pre-treated MoDCs was observed ( Supplementary Figures 9A, B ). These data largely supported the notion that fibrillar α-syn can be encapsulated in autophagic components which likely allowed the processing of α-syn through autophagy. However, this happens to a lesser extent in MoDCs but can be alleviated by Celastrol. Lastly, the level of active GTP forms of Rab5 and Rab7 were upregulated by Celastrol pre-treatment ( Figures 6G–J ), in corroboration with the above data. Together, Celastrol not only induced autophagy by upregulating Beclin1 expression, reducing p62 expression and increasing LC3 conversion, but it can also trigger the amphisomal pathway with increased functional Rab5 and Rab7 proteins to interact with autophagic components for trafficking of α-syn towards the lysosomal-mediated processing.