Journal: The Journal of Clinical Investigation

Article Title: N-acetyl- l -leucine lowers α -synuclein levels and improves synaptic function in Parkinson’s disease models

doi: 10.1172/JCI196137

Figure Lengend Snippet: ( A ) Representative Western blot showing pS129-syn and total α-syn (Syn) levels following 30 days of treatment with increasing concentrations of NALL in the Triton-soluble fraction of GBA1 L444P mutant dopaminergic neurons. β-III-tubulin and GAPDH served as loading controls. ( B ) Quantification of pS129-syn (top) and total Syn (bottom) signals in A , normalized to β-III-tubulin and expressed relative to the 0 mM (DMSO) group ( n = 3–4 independent experiments; 1-way ANOVA). ( C ) Western blot of pS129-syn and total Syn in the Triton-insoluble fraction of GBA1 L444P mutant neurons following NALL treatment; total protein staining served as a loading control. ( D ) Quantification of pS129-syn (top) and total Syn (bottom) signals in C , normalized to total protein and expressed relative to the 0 mM (DMSO) group ( n = 3–4 independent experiments; 1-way ANOVA). ( E – H ) Similar analyses performed in GBA1 N370S mutant dopaminergic neurons. ( E and G ) Representative blots of soluble ( E ) and insoluble ( G ) fractions with corresponding quantifications ( F and H ), normalized and expressed relative to the 0 mM (DMSO) group ( n = 3 independent experiments; 1-way ANOVA). ( I ) (Top) Schematic of the experimental design showing NALL treatment (10 mM) of GBA1 L444P mutant dopaminergic neurons at day 120 for 7, 14, or 21 days. (Bottom) Western blot of pS129-syn in the Triton-soluble fraction with or without NALL treatment. β-III-tubulin and GAPDH were used as loading controls. ( J ) Quantification of pS129-syn levels in I , normalized to β-III-tubulin and expressed relative to the nontreated (NT; DMSO) group ( n = 3 independent experiments; 2-way ANOVA). ( K ) Western blot of pS129-syn in the Triton-insoluble fraction of GBA1 L444P neurons with or without NALL treatment; total protein staining was used as a loading control. ( L ) Quantification of pS129-syn in K , normalized to total protein and expressed relative to the NT (DMSO) group ( n = 3 independent experiments; 2-way ANOVA). All data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.005.

Article Snippet: Antibodies used for immunoblotting include anti-pS129-syn antibody (Cell Signaling Technology: 23706S), anti-SYP antibody (MilliporeSigma: ab9272), anti-α-syn antibody (Santa Cruz Biotechnology: sc-7011-R), anti-β-III-tubulin antibody (BioLegend: 801202), anti-SYNJ1 antibody (LsBio: 65169), anti-parkin antibody (Santa Cruz Biotechnology: sc-32282), anti-HTRA1 (R&D Systems: MAB2916), anti-HTRA1 (Proteintech: 55011-1-AP), anti-caspase 3 (Cell Signaling Technology: 9662), anti-cleaved caspase-3 (Cell Signaling Technology: 9661S), anti-DAT antibody (Santa Cruz Biotechnology: sc-32259), anti-GAPDH antibody (MilliporeSigma: MAB374), Goat Anti-Rat IgG (H+L) (Peroxidase AffiniPure: 112-035-062), Goat Anti-Rabbit IgG (H+L) (Peroxidase-AffiniPure: 111-035-144), and Goat Anti-Mouse IgG (H+L) (Peroxidase-AffiniPure: 115-035-146).

Techniques: Western Blot, Mutagenesis, Staining, Control

Journal: The Journal of Clinical Investigation

Article Title: N-acetyl- l -leucine lowers α -synuclein levels and improves synaptic function in Parkinson’s disease models

doi: 10.1172/JCI196137

Figure Lengend Snippet: ( A ) Heatmap showing proteins significantly altered (adjusted P < 0.05) by 30 days of NALL treatment in GBA1 L444P mutant neurons ( n = 3 biological replicates; 3,801 total proteins). ( B ) Volcano plot of protein changes induced by 10 mM NALL versus nontreated (NT) controls. HTRA1 (green) and other significantly altered proteins. Dashed line, adjusted P = 0.05 threshold (1-way ANOVA with Benjamini-Hochberg correction). ( C – H ) Representative Western blots and corresponding quantification of HTRA1 and pS129-syn in total lysates ( C and D ), Triton-soluble fractions ( E and F ), and Triton-insoluble fractions ( G and H ) of GBA1 L444P neurons treated with increasing NALL concentrations for 30 days. β-III-tubulin, GAPDH, or total protein staining served as loading controls ( n = 3 independent experiments). ( I – K ) Western blot analysis ( I ) and quantification of HTRA1 ( J ) and pS129-syn ( K ) in GBA1 L444P neurons following HTRA1 knockdown (KD-1 and KD-2) with or without 10 mM NALL. Data were normalized to GAPDH or β-III-tubulin and expressed relative to NT-scramble controls ( n = 4 independent experiments). Statistical significance was determined by 1-way ANOVA with Benjamini-Hochberg correction ( B ), 1-way ANOVA ( D , F , H , and J right), or 2-way ANOVA ( J left and K ). Data represent mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001.

Article Snippet: Antibodies used for immunoblotting include anti-pS129-syn antibody (Cell Signaling Technology: 23706S), anti-SYP antibody (MilliporeSigma: ab9272), anti-α-syn antibody (Santa Cruz Biotechnology: sc-7011-R), anti-β-III-tubulin antibody (BioLegend: 801202), anti-SYNJ1 antibody (LsBio: 65169), anti-parkin antibody (Santa Cruz Biotechnology: sc-32282), anti-HTRA1 (R&D Systems: MAB2916), anti-HTRA1 (Proteintech: 55011-1-AP), anti-caspase 3 (Cell Signaling Technology: 9662), anti-cleaved caspase-3 (Cell Signaling Technology: 9661S), anti-DAT antibody (Santa Cruz Biotechnology: sc-32259), anti-GAPDH antibody (MilliporeSigma: MAB374), Goat Anti-Rat IgG (H+L) (Peroxidase AffiniPure: 112-035-062), Goat Anti-Rabbit IgG (H+L) (Peroxidase-AffiniPure: 111-035-144), and Goat Anti-Mouse IgG (H+L) (Peroxidase-AffiniPure: 115-035-146).

Techniques: Mutagenesis, Western Blot, Staining, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: N-acetyl- l -leucine lowers α -synuclein levels and improves synaptic function in Parkinson’s disease models

doi: 10.1172/JCI196137

Figure Lengend Snippet: ( A – D ) Representative Western blots and quantification of pS129-syn, parkin, and HTRA1 in LRRK2 R1441C mutant dopaminergic neurons treated with increasing NALL concentrations for 14 days. Analysis was performed in Triton-soluble ( A and B ) and Triton-insoluble ( C and D ) fractions. ( E – H ) Western blot and quantification of pS129-syn, parkin, and HTRA1 in soluble and insoluble fractions of LRRK2 G2019S mutant neurons treated with NALL. ( I – L ) Western blot and quantification of pS129-syn, parkin, and HTRA1 in VPS35 D620N mutant dopaminergic neurons treated with or without NALL. ( M – P ) Western blot and quantification of pS129-syn, parkin, and HTRA1 in dopaminergic neurons derived from patients with sporadic PD treated with or without NALL. Data are expressed relative to the 0 mM (DMSO) or nontreated group ( n = 3–4 independent experiments). Statistical significance was determined by 1-way ANOVA ( B , D , F , and H ) or Student’s t test ( J , L , N , and P ). All data represent mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

Article Snippet: Antibodies used for immunoblotting include anti-pS129-syn antibody (Cell Signaling Technology: 23706S), anti-SYP antibody (MilliporeSigma: ab9272), anti-α-syn antibody (Santa Cruz Biotechnology: sc-7011-R), anti-β-III-tubulin antibody (BioLegend: 801202), anti-SYNJ1 antibody (LsBio: 65169), anti-parkin antibody (Santa Cruz Biotechnology: sc-32282), anti-HTRA1 (R&D Systems: MAB2916), anti-HTRA1 (Proteintech: 55011-1-AP), anti-caspase 3 (Cell Signaling Technology: 9662), anti-cleaved caspase-3 (Cell Signaling Technology: 9661S), anti-DAT antibody (Santa Cruz Biotechnology: sc-32259), anti-GAPDH antibody (MilliporeSigma: MAB374), Goat Anti-Rat IgG (H+L) (Peroxidase AffiniPure: 112-035-062), Goat Anti-Rabbit IgG (H+L) (Peroxidase-AffiniPure: 111-035-144), and Goat Anti-Mouse IgG (H+L) (Peroxidase-AffiniPure: 115-035-146).

Techniques: Western Blot, Mutagenesis, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: N-acetyl- l -leucine lowers α -synuclein levels and improves synaptic function in Parkinson’s disease models

doi: 10.1172/JCI196137

Figure Lengend Snippet: ( A ) Western blots showing pS129-syn, parkin, and HTRA1 in the Triton-soluble fraction of the substantia nigra from LRRK2 R1441C mice treated with NALL (NALL) or vehicle (NT). β-III-tubulin and GAPDH were used as loading controls. ( B ) Quantification of fold-changes in pS129-syn (left), parkin (middle), and HTRA1 (right) following NALL treatment shown in A . Data represent average pS129-syn levels (normalized to β-III-tubulin), parkin (normalized to GAPDH), and HTRA1 (normalized to GAPDH), expressed relative to the NT (vehicle-only) group ( n = 9 mice; t test). ( C ) Western blots showing pS129-syn and HTRA1 in the Triton-insoluble fraction of LRRK2 R1441C mouse substantia nigra with or without NALL treatment. Total protein staining was used as a loading control. ( D ) Quantification of fold-changes in pS129-syn and HTRA1 from C . Data represent average pS129-syn and HTRA1 (normalized to total protein) levels relative to the NT (vehicle-only) group ( n = 9 mice; t test). Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies used for immunoblotting include anti-pS129-syn antibody (Cell Signaling Technology: 23706S), anti-SYP antibody (MilliporeSigma: ab9272), anti-α-syn antibody (Santa Cruz Biotechnology: sc-7011-R), anti-β-III-tubulin antibody (BioLegend: 801202), anti-SYNJ1 antibody (LsBio: 65169), anti-parkin antibody (Santa Cruz Biotechnology: sc-32282), anti-HTRA1 (R&D Systems: MAB2916), anti-HTRA1 (Proteintech: 55011-1-AP), anti-caspase 3 (Cell Signaling Technology: 9662), anti-cleaved caspase-3 (Cell Signaling Technology: 9661S), anti-DAT antibody (Santa Cruz Biotechnology: sc-32259), anti-GAPDH antibody (MilliporeSigma: MAB374), Goat Anti-Rat IgG (H+L) (Peroxidase AffiniPure: 112-035-062), Goat Anti-Rabbit IgG (H+L) (Peroxidase-AffiniPure: 111-035-144), and Goat Anti-Mouse IgG (H+L) (Peroxidase-AffiniPure: 115-035-146).

Techniques: Western Blot, Staining, Control

Journal: World Journal of Gastrointestinal Oncology

Article Title: Pancreatic neuroendocrine microtumors in the elderly: A retrospective study using cadaveric pancreatic tissue

doi: 10.4251/wjgo.v17.i12.113198

Figure Lengend Snippet: Histopathological changes of pancreatic neuroendocrine microtumors in Case 4. A: Hematoxylin and eosin staining of pancreatic neuroendocrine microtumors (PNEMTs) of Case 4; B: Immunostaining of PNEMTs in A with synaptophysin (Syn) antibody; C: Immunofluorescence staining of PNEMTs with glucagon (GLU) antibody; D: GLU (green) with insulin (red) double immunofluorescence staining. White asterisk in A: The tumors exhibited an organoid-like architecture, with tumor cells arranged in a trabecular pattern; black asterisk in A: The surrounding pancreatic parenchyma appeared largely normal. Black arrows in A: Indicate a slight membrane structure between the PNEMTs and the pancreatic tissue. Black arrowheads in A: Acinar ductal metaplasia; White arrows in C: GLU-positive cells in the islets of Langerhans; Black and white arrows in D: Insulin-positive cells in PNEMTs and islets of Langerhans, respectively. GLU: Glucagon; Syn: Synaptophysin; Ins: Insulin.

Article Snippet: The primary antibodies used were polyclonal guinea pig anti-human insulin antibody (ready-to-use, IR00261-2J; Dako, Tokyo, Japan), rabbit anti-GLU antibody (1:100, ab92517; Abcam, Tokyo, Japan), mouse anti-human chromogranin A antibody (1:1000, AMAb90525; Atlas Antibodies AB, Stockholm, Sweden), rabbit anti-human synaptophysin (Syn) antibody (1:100, HPA002858; Atlas Antibodies AB, Stockholm, Sweden), and rabbit anti-Ki-67 antibody (1:100, ab16667; Abcam, Tokyo, Japan) diluted in PBS supplemented with 3% bovine serum albumin.

Techniques: Staining, Immunostaining, Immunofluorescence, Double Immunofluorescence Staining, Membrane

Journal: Translational Neurodegeneration

Article Title: Attenuating α-synuclein pathology in mice with in situ engineered astrocytes

doi: 10.1186/s40035-025-00518-0

Figure Lengend Snippet: Expression of CAR-A and effect on α-synO phagocytosis and digestion. a Design of the CAR-expression plasmid. CAR was expressed in fusion with 3A scFv and enhanced green fluorescent protein (EGFP) under the control of the astrocyte-specific promotor GfaABC1D. SP, signal peptide; Poly(A), polyadenylation signal; ORI, origin of replication; KanR, kanamycin resistance gene. b Representative image of CAR expression on an astrocyte. The co-localization of 3A, MerTK and EGFP was assessed by confocal microscopy. Scale bars, 10 μm. c Flow cytometry analysis of the binding of CAR-A and ns-CAR-A to α-syn monomers and oligomers (α-synOs). The astrocytes were transfected with CAR or ns-CAR lipoplexes for 48 h. After 2 h-incubation with 1 μmol/L α-syn monomers (α-syn) or α-synOs, cells were stained with PE-labeled anti-α-syn antibody. d PE fluorescence in EGFP-positive astrocytes. n = 3 independent experiments. e Flow cytometry analysis of the amount of α-synO engulfed by CAR-A, ns-CAR-A and NC-A in the presence of different α-synO concentrations. n = 3 independent experiments. f Representative images depicting the phases of engulfment and digestion of α-synO by CAR-A. CAR-A was treated with 1 μmol/L α-synO, and the medium was changed after 1 h incubation. α-SynO and Lamp1 in CAR-A were stained with respective antibodies at different time points and imaged by confocal microscopy. Scale bars, 5 μm. g The kinetic curves of α-synO digestion in CAR-A, ns-CAR-A and NC-A. n = 3 independent experiments. h Statistical analysis of the proportion of α-syn colocalized with Lamp1 in digestion stage in ( f ) by Image J. n = 4 independent experiments. i Intracellular α-syn in Triton X-100-soluble and -insoluble fraction detected by Western blotting at different time points post astrocytic phagocytose of α-synOs. β-actin was used as a control. j Quantification of α-syn ( i ) using Image J. n = 3 independent experiments. k Representative images depicting the binding of ns-CAR-A, NC-A and CAR-A to α-syn monomers and oligomers. Scale bars, 5 μm. Data are mean ± S.E.M. One-way ANOVA ( d ) or Two-way ANOVA ( e ) followed by Tukey’s multiple comparison test was used for statistical analysis. * P < 0.05, ** P < 0.01, **** P < 0.0001 indicate significance compared to respective groups

Article Snippet: Primary antibodies for the following proteins were used: MerTK (Abcam, #ab95925, RRID: AB_10863559, 1:100), 3A (monoclonal antibody, prepared by our laboratory, 1:100), α-syn (Abcam, #ab138501, RRID: AB_2537217, 1:100; BD biosciences, Franklin Lakes, NJ, #610787, RRID: AB_398108, 1:100), pS129 α-syn (Cell Signaling Technology, Danvers, MA, #23706, RRID: AB_2798868, 1:100), Lamp1 (Abcam, #ab24170, RRID: AB_775978, 1:100), ionized calcium binding adaptor molecule 1 (Iba-1) (GeneTex, Irvine, TX, #GTX100042, RRID: AB_1240434, 1:100); microtubule-associated protein 2 (MAP2) (Abcam, #ab32454, RRID: AB_776174, 1:100), glial fibrillary acidic protein (GFAP) (Cell Signaling Technology, #3670S, RRID: AB_561049, 1:100; Affinity, Nanjing, China, #DF6040, AB_2838012, 1:100; Abcam, #ab302644, 1:100), C3 (Abcam, #ab97462, RRID: AB_10679468, 1:100), and S100β (SinoBiological, Beijing, China, #100508-MM30, 1:100).

Techniques: Expressing, Plasmid Preparation, Control, Confocal Microscopy, Flow Cytometry, Binding Assay, Transfection, Incubation, Staining, Labeling, Fluorescence, Western Blot, Comparison

Journal: Translational Neurodegeneration

Article Title: Attenuating α-synuclein pathology in mice with in situ engineered astrocytes

doi: 10.1186/s40035-025-00518-0

Figure Lengend Snippet: CAR-A reduced α-syn levels in PFF-seeded A53T mice. a Representative images depicting α-synO engulfment in different experimental groups in the striatum. Scale bars, 50 μm (upper four panels and eight small images below them) and 10 μm (lower four magnifications of zoomed areas and eight small images below them). b Quantification of the engulfed α-syn in GFAP-positive cells using Image J. n = 9 biologically independent animals. c , d The levels of α-synO specifically detected by 3A in brainstem were assessed by immunoblotting ( c ) and quantified by Image J ( d ). n = 6 biologically independent animals. e p-MerTK levels in the striatum of different groups assessed by western blotting. MerTK was used as a control. f Quantification of p-MerTK levels in ( e ) using Image J. n = 5 biologically independent animals. g , h Total α-syn levels and pS129 α-syn levels in Triton X-100-insoluble ( g ) and -soluble ( h ) fractions of brainstem assessed by western blotting. β-Actin was used as a control. i , j Quantification of total α-syn levels and pS129 α-syn levels in Triton X-100-insoluble ( i ) and -soluble ( j ) fractions of brainstem using Image J. n = 6 biologically independent animals. k , l The levels of pS129 α-syn in the substantia nigra were monitored by immunostaining ( k ), and quantified by Image J ( l ). Scale bar, 200 μm (low-magnification images) and 50 μm (high-magnification images), respectively. n = 9 biologically independent animals. Data are mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison tests was conducted for statistical analyses. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 indicate significance compared to respective groups. NS, not significant

Article Snippet: Primary antibodies for the following proteins were used: MerTK (Abcam, #ab95925, RRID: AB_10863559, 1:100), 3A (monoclonal antibody, prepared by our laboratory, 1:100), α-syn (Abcam, #ab138501, RRID: AB_2537217, 1:100; BD biosciences, Franklin Lakes, NJ, #610787, RRID: AB_398108, 1:100), pS129 α-syn (Cell Signaling Technology, Danvers, MA, #23706, RRID: AB_2798868, 1:100), Lamp1 (Abcam, #ab24170, RRID: AB_775978, 1:100), ionized calcium binding adaptor molecule 1 (Iba-1) (GeneTex, Irvine, TX, #GTX100042, RRID: AB_1240434, 1:100); microtubule-associated protein 2 (MAP2) (Abcam, #ab32454, RRID: AB_776174, 1:100), glial fibrillary acidic protein (GFAP) (Cell Signaling Technology, #3670S, RRID: AB_561049, 1:100; Affinity, Nanjing, China, #DF6040, AB_2838012, 1:100; Abcam, #ab302644, 1:100), C3 (Abcam, #ab97462, RRID: AB_10679468, 1:100), and S100β (SinoBiological, Beijing, China, #100508-MM30, 1:100).

Techniques: Western Blot, Control, Immunostaining, Comparison