Fig 2: Purity and specificity of 4B1 toward WT–α-syn aggregates. (A) Fifty nanograms of recombinant WT–, pS129–, or S129A–α-syn was loaded on 15% SDS gels and transferred to nitrocellulose membranes for Western blotting for 4B1, in-house pS129, and Syn-1. (B, i) Analysis of filter retardation using monomeric– (M) or fibrillar– (F) α-syn coated on nitrocellulose membranes and detected with 4B1, Syn-1, and Syn-O2. Reactivity of 4B1 toward different α-syn aggregates including fibrils, ONE, or HNE (ii) 4B1 preincubated with monomers or ONE-oligomers and tested against precoated monomeric–α-syn in inhibition ELISA. (iii) Sandwich-ELISA showing the reactivity of 4B1 toward monomers or different α-syn aggregates. (iv) Specificity of 4B1 toward WT–α-syn aggregates. PFFs and fibrils were treated for 6 h with increasing concentrations of denaturing urea and SDS, with 4B1 detecting the aggregated forms of α-syn. (v) Filter retardation assessment for 4B1 reactivity toward α-syn fibrils. F11 detects α- or β-syn, whereas the antibody E20 detects γ-syn. Filter retardation analysis of 4B1 reactivity toward human (H–α-syn) or mouse (M–α-syn) fibrils. (C) Ten micrograms of brain lysates from human SNCA transgenic (lanes 1 and 2) and SNCA-null mice (lanes 3 and 4) were immunoblotted using antibodies specific to WT–α-syn (4B1) and total α-syn (Syn-1). Recombinant pS129–α-syn (rpS129–α-syn, lane 5) and recombinant α-syn (rα-syn, lane 6) proteins were loaded (50 ng) as positive controls. Reimmunoblotting with β-actin antibody was performed to normalize the amount of loaded proteins. (D) Immunofluoresence images of striatal brain sections from adult male α-syn KO mice, showing no 4B1 or pS129 immunoreactivity in the tyrosine hydroxylase (TH, red) -positive dopaminergic fibers (63× magnification). (Scale bar, 50 μm.)

Fig 3: Effects of Anesthesia/Surgery/ICU on expression of synuclein alpha, neurotrophic receptor tyrosine kinase 1 and syntaxin 1a genes. (A) Snca α, Ntrk1, and STX1a mRNA levels were significantly impaired in A/S/I mice relative to controls following ICU environment (–3.785, –1.498, and –2.268 fold decrease, respectively). Hippocampal total RNA was extracted from A/S/I animals (pooled N = 3) and controls (pooled N = 3). (B–E) Protein levels of STX1a (B: P < 0.012), Ntrk1 (C: P = 0.039), and Snca α (D: P = 0.017) were significantly decreased in A/S/I mice relative to controls. Snca phosphorylated at serine residue 129 was also significantly down-regulated in insult-exposed mice (E: P = 0.008). Two-tailed unpaired t-test (N = 7 A/S/I and N = 7 control mice). STX1a, syntaxin 1a; NtrK1, neurotrophic tropomyosin receptor kinase 1; Snca α, synuclein alpha; p-Snca α, synuclein alpha phosphorylated at serine residue 129. ∗P < 0.05 and ∗∗P < 0.01, respectively.

Fig 4: Exacerbated α-synuclein accumulation in apoE4 cerebral organoids and postmortem brains from APOE4 carriers. Cerebral organoids were generated from iPSC lines carrying APOE ε3/ε3 (APOE3/3) or ε4/ε4 (APOE4/4) genotype and subjected to analyses at Day 90. a–d, Amounts of αSyn (b), p-αSyn (c) and apoE (d) in the RIPA fraction of the iPSC-derived cerebral organoids were quantified by Western blotting. e–j, Amounts of αSyn (f), p-αSyn (g) and apoE (h) in the SDS fraction of the iPSC-derived cerebral organoids were quantified by Western blotting. ApoE, αSyn, and p-αSyn levels were normalized to β-actin levels. Spearman correlation analyses for apoE vs. αSyn (i) and apoE vs. p-αSyn (j) in SDS fraction are shown with the correlation coefficient (r) and the correlation p value. Experiments were repeated in two independent differentiation batches. Lysates of 3 cerebral organoids from each line were analyzed as one sample. All data are expressed as mean ± SEM (N = 5 lines/each). k–l, Postmortem brain sections from the superior temporal cortex of Lowy body dementia (LBD) subjects with or without APOE4 were immunostained with apoE antibody and anti-αSyn NACP98 antibody for Lewy body. Representative images for the deposition of apoE in NACP-positive Lewy bodies are shown (k). Scale bar: 20 µm. The colocalization of apoE with NACP-positive Lewy bodies was quantified by Image J (l). All data are expressed as mean ± SEM (N = 17 patients/each). Mann–Whitney U tests were performed to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: Treatment with Glucosylceramide synthase inhibitor decreases mTORC1 activity and increases TFEB expression in GBA1 mutant PD neurons. (A) Representative immunofluorescence images of WT control, gene-corrected, and GBA1 mutant neurons co-labeled with anti-p-mTOR (green) and anti-MAP2 (red). Also shown is nuclear DAPI. GBA1 mutant neurons were either untreated or treated with 5 μM of the lipid substrate reduction compound Genz-123346 (GZ) for 3 days. Scale bar = 50 μm. Bar graph represents mean p-mTOR fluorescence signal intensity ±SEM. Data were collected at least 50 cells per group, assayed in 3–4 different fields in 1–2 independent experiments. ****p<0.0001 (WT vs. PD4), ***p = 0.0004 (PD4 vs. gene-corrected), and ****p < 0.0001 (PD4 vs. PD4 GZ) as assessed by One-way ANOVA. (B) Representative immunofluorescence images of WT control and GBA1 mutant neurons co-labeled with antibodies to p-RPS6 (green) and MAP2 (red). Cells were either untreated or treated with GZ. Scale bar = 50 μm. Bar graph represent mean p-RPS6 fluorescence signal intensity. Data were collected from >100 cells per group, assayed in 3 different fields per experiment in 3 independent experiments. Error bars = SEM, *p = 0.01 and **p = 0.006 between the indicated groups as assessed by One-way ANOVA. (C) Western blot analysis for p-RPS6 levels in WT control and GBA1 mutant PD DNCs. Also shown is β-Actin loading control. Cells were either untreated or treated with 5 μM GZ for 3–5 days. Bar graph represents folds relative to untreated control cells. Data represent average (from PD1-GBAWT/N370S, PD2-GBAWT/N370S and PD3-GBAWT/N370S combined) ± SEM, n = 3–5 per group. *p = 0.01 (between WT and PD) and *p = 0.02 (between PD and PD with GZ) as assessed by One-way ANOVA. (D) Representative immunofluorescence images of WT control and GBA1 mutant neurons co-labeled with antibodies to TFEB (green) and MAP2 (red). Also shown is nuclear DAPI. GBA1 mutant neurons were either untreated or treated with 5 μM GZ for 3 days. Scale bar = 25 μm. Bar graph to the left represents mean TFEB fluorescence signal intensity. Data were collected from >50 cells per group, assayed in 3 different fields in 2–3 independent experiments. Error bars = SEM, ****p < 0.0001 and **p = 0.006 between the indicated groups as assessed by One-way ANOVA. (E) Representative immunofluorescence images of WT control and GBA1 mutant neurons co-labeled with antibodies to TFEB (green) and MAP2 (red). Also shown is nuclear DAPI. GBA1 mutant neurons were either untreated or treated with 5 μM GZ for 3 days. Arrows point to TFEB fluorescence signal colocalization with nuclear DAPI. Scale bar = 50 μm.