mouse α syn  (Proteintech)

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: Antibodies used for in vitro investigations.

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: In Vitro

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: Body mass evolution during 35 days of daily treatment with vehicle or AZP2006 post-lesion (A). Representative IHC images TH (red)/α-syn (green)/Iba1 (purple) (B). IHC analysis shows the total number of TH(+) neurons (C), area of α-syn accumulation in TH(+) neurons (D) and the number of Iba1(+) microglia in the SNpc (E) expressed as % of sham. Raw values for sham (i.e., 100%); TH(+) neurons = 65 ± 1.6; α-syn area/TH(+) neuron = 168 ± 9.4 µm 2 ; Iba1(+) cells = 53 ± 2.8. Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ## p < 0.01 or #### p < 0.0001 from sham; **p < 0.01; ***p < 0.001 and ****p < 0.0001 from α-syn+vehicle injury, (n = 7 mice / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis of cultures shows the total number of TH(+) neurons (A,E), the length of their neurite network (B,F), the area of cytoplasmic α-syn staining per TH(+) neuron (C,G) and the area of the post-synaptic marker PSD95 per TH(+) neuron (D and H), expressed as % of control (CTR). Raw values for CTR (i.e., 100%); sum TH(+) neurons per well, i.e., per biological replicate = 27 ± 1.0; neurite network = 3540 ± 127 µm; α-syn area/TH(+) neuron = 201 ± 8.9 µm 2 ; PSD95 area/TH(+) neuron = 1829 ± 105 µm 2 . Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ## p < 0.01; ### p < 0.001 or #### p < 0.0001 from CTR (control) or from anti-PGRN Ab and *p < 0.05 or ****p < 0.0001 from 6OHDA or from 6OHDA + anti-PGRN Ab; (n = 4-6 / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Staining, Marker, Control

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis of cultures shows the total number of TH(+) neurons (A,E), the length of their neurite network (B,F), the area of cytoplasmic α-syn per TH(+) neuron (C,G) and lysosome burden as area of LAMP2(+) vesicles per TH(+) neuron (D and H), expressed as % of control (CTR). Raw values for MPP + & Rotenone CTR (i.e., 100%); sum TH(+) neurons per well, i.e., per biological replicate = 18 ± 0.5 & 26 ± 1; neurite network = 889 ± 57 & 929 ± 24 µm; α-syn area/TH(+) neuron = 29 ± 2.1 & 28 ± 1 µm 2 ; ratio LAMP2/TH(+) = 10 ± 0.3 & 10 ± 0.5, respectively. Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. #### p < 0.0001 from CTR and *p < 0.05, **p < 0.01; ***p < 0.001 or ****p < 0.0001 from MPP + or Rotenone treatment, (n = 4-6 / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Control

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis of cultures shows the total number of TH(+) neurons (A), the length of their neurite network (B), the area of cytoplasmic α-syn staining per neuron (C), the area of LAMP2(+) staining per TH(+) neuron (D), the α-syn accumulation in lysosomes as α-syn/LAMP2 colocalization area (E), autophagosomes as LC3b(+) vesicles per TH(+) neuron (F), autolysosomes as LC3b(+) vesicles colocalized with LAMP2 area in TH(+) neurons (G), and functional lysosomes expressed as area of PhenoVue 577(+) staining per SH-SY5Y cell (H), expressed as % of control (CTR). Raw values for CTR (i.e., 100%); sum TH(+) neurons per well, i.e., per biological replicate = 32 ± 1.3; neurite network = 1054 ± 60.6 µm; α-syn area/TH(+) neuron = 25.2 ± 2.8 µm 2 ; LAMP2 area/TH(+) neurons = 17.9 ± 1.5 µm 2 ; α-syn area/LAMP2/TH(+) neuron = 4.4 ± 0.3 µm 2 ; LC3b area/TH(+) neurons = 27 ± 1.7 µm 2 ; LAMP2/LC3b area/TH(+) neurons = 7.8 ± 0.5 µm 2 ; PhenoVue 577(+) staining/SH-SY5Y cell = 67 ± 3.5 µm 2 . Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ### p < 0.001 or #### p < 0.0001 from CTR; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 from the α-syn condition, (n = 5-6).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Staining, Functional Assay, Control

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis of cultures shows the total number of TH(+) neurons (A) and the length of their neurite network neurons (B) and microglia activation as area of OX-41(+) staining (C), expressed as % of control. Raw values for control (CTR) (i.e., 100%); sum TH(+) neurons per well, i.e., per biological replicate = 81 ± 4.0; neurite network = 2332 ± 112 µm; OX-41(+) area/field = 1421 ± 67 µm 2 . Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ### p < 0.001 or #### p < 0.0001 from CTR; **p < 0.01, ***p < 0.001 and ****p < 0.0001 from α-syn injury, (n = 4-6 / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Activation Assay, Staining, Control

Journal: PLOS One

Article Title: AZP2006 in Parkinson’s disease models: A promising new neuroprotective treatment involving lysosomal homeostasis and the Progranulin/Prosaposin complex

doi: 10.1371/journal.pone.0359269

Figure Lengend Snippet: ICC analysis shows the total number of cells (A, D), the area of cytoplasmic α-syn staining per cell (B,E) and lysosomal burden as area of LAMP2(+) vesicles per cell (C,F), expressed as % of control (CTR = PGRN KO alone in D-F). Raw values for PGRN KO CTR (i.e., 100%); cells/image field = 140 ± 3.9; α-syn area/cell = 38.1 ± 2.8 µm 2 ; LAMP2(+) area/cell = 51 ± 2.8 µm 2 . Each bar represents the mean ± SEM, one-way ANOVA followed by Fisher’s test. ## p < 0.01 or #### p < 0.0001 from CTR and *p < 0.05, **p < 0.01; ***p < 0.001 or ****p < 0.0001 from MPP + (n = 4-6 / group).

Article Snippet: α-syn , Guinea pig , Polyclonal , Alomone , APZ-035-GP , AB_2925147 , 3.

Techniques: Analysis, Staining, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Clemastine alleviates Parkinson’s disease pathology by activating the AMPK/mTOR pathway and enhancing autophagic flux

doi: 10.3389/fnagi.2026.1915475

Figure Lengend Snippet: Study and experimental flow chart. This Figure illustrates the overall experimental design and workflow of the study. The study integrates bioinformatic analyses with in vitro and in vivo experimental validations. Bioinformatic approaches included the single-cell transcriptome analysis of Parkinson’s disease (PD) patient midbrains to identify differentially expressed genes (DEGs) in dopaminergic neurons (DaNs) and cell type alterations, molecular docking between clemastine (CLE) and 22,618 proteins to identify potential targets, and correlation analysis between Braak staging and autophagy gene expression. The intersection of CLE targets and PD-related DEGs was used to predict the CLE’s mechanism of action, focusing on the AMPK/mTOR pathway and autophagy. These predictions were tested experimentally using an MPP + -induced PC12 cell model and a chronic MPTP-induced mouse model of PD. Endpoints assessed included protein expression (western blot and immunofluorescence), autophagic flux, α-synuclein (α-syn) and phosphorylated α-syn (p-α-syn) levels, dopaminergic neuron survival, motor function (rotarod test), and non-motor function (fecal pellet analysis). The integrative approach aims to demonstrate that CLE alleviates PD pathology by activating the AMPK/mTOR pathway, enhancing autophagic flux, and promoting the clearance of pathological α-syn. PD, Parkinson’s disease; CLE, Clemastine; MPTP, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP + , 1-methyl-4-phenylpyridinium; DEGs, differentially expressed genes; DaNs, dopaminergic neurons; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; α-syn, α-synuclein; p-α-syn: phosphorylated α-synuclein.

Article Snippet: Proteins were transferred to PVDF membranes at 30 V for 90 min. Membranes were blocked with 5% non-fat milk in PBST for 1 h and incubated with primary antibodies for 2 h at room temperature: 1:50,000 mouse GAPDH (60004-1-Ig, Proteintech), 1:2,000 mouse p-mTOR (Ser2448) (67778-1-Ig, Proteintech), 1:5,000 mouse t-mTOR (66888-1-Ig, Proteintech), 1:1000 rabbit p-AMPK (Thr183/172) (ab133448, Abcam), 1:1000 mouse p62 (66184-1-Ig, Proteintech), 1:1,000 rabbit LC3-I/II (orb411847, Biorbyt), 1:5,000 mouse P62 (66184-1-Ig, Proteintech), 1:500 mouse α-syn (66412-1-Ig, Proteintech).

Techniques: In Vitro, In Vivo, Single Cell, Analysis, Docking Assay, Gene Expression, Expressing, Western Blot, Immunofluorescence, Pathology

Journal: Frontiers in Aging Neuroscience

Article Title: Clemastine alleviates Parkinson’s disease pathology by activating the AMPK/mTOR pathway and enhancing autophagic flux

doi: 10.3389/fnagi.2026.1915475

Figure Lengend Snippet: CLE reduces α-synuclein protein levels and improves cell survival, effects inhibited by dorsomorphin (DOR). (A) Western blot (WB) and (B) The quantification of α-synuclein (α-syn) protein levels in PC12 cells from CTRL, MPP + , CLE, and MPP + +CLE groups. GAPDH is shown as a loading control. (C) Cell survival rate in CTRL, MPP + , and MPP + +CLE groups. (D, G) The WB analysis of p-AMPK (Thr172), t-AMPK, p-mTOR (Ser2448), and t-mTOR protein levels in CTRL, DOR, CLE, and DOR+CLE groups. (E, F, H) The Quantification of the indicated protein ratios. Data are presented as mean ± SEM. Tukey’s multiple comparison test was performed after one-way ANOVA. * P < 0.05, ** P < 0.01 *** P < 0.001; ns, not significant. CLE, clemastine; α-syn, α-synuclein; CTRL, control; WB, western blot; DOR, dorsomorphin; ANOVA, analysis of variance; SEM, standard error of the mean. n = 5 independent experiments.

Article Snippet: Proteins were transferred to PVDF membranes at 30 V for 90 min. Membranes were blocked with 5% non-fat milk in PBST for 1 h and incubated with primary antibodies for 2 h at room temperature: 1:50,000 mouse GAPDH (60004-1-Ig, Proteintech), 1:2,000 mouse p-mTOR (Ser2448) (67778-1-Ig, Proteintech), 1:5,000 mouse t-mTOR (66888-1-Ig, Proteintech), 1:1000 rabbit p-AMPK (Thr183/172) (ab133448, Abcam), 1:1000 mouse p62 (66184-1-Ig, Proteintech), 1:1,000 rabbit LC3-I/II (orb411847, Biorbyt), 1:5,000 mouse P62 (66184-1-Ig, Proteintech), 1:500 mouse α-syn (66412-1-Ig, Proteintech).

Techniques: Western Blot, Control, Analysis, Comparison

Journal: Frontiers in Aging Neuroscience

Article Title: Clemastine alleviates Parkinson’s disease pathology by activating the AMPK/mTOR pathway and enhancing autophagic flux

doi: 10.3389/fnagi.2026.1915475

Figure Lengend Snippet: CLE increases AMPK phosphorylation, decreases mTOR phosphorylation at Ser2448, enhances autophagy, and reduces α-synuclein levels in the mouse midbrain. (A) The WB analysis of p-AMPK (Thr172), t-AMPK, p-mTOR (Ser2448), and t-mTOR protein levels in the midbrain of mice from CTRL, MPTP, and MPTP+CLE groups. GAPDH is shown as a loading control. (B) The quantification of p-mTOR/t-mTOR levels. (C) The quantification of p-AMPK/t-AMPK levels. (D–F) The WB analysis of p62, LC3-I/II, and α-syn protein levels. (G–I) The quantification of p62, LC3-II/LC3-I ratio, and α-syn protein levels. Data are presented as mean ± SEM. Tukey’s multiple comparison test was performed after one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. CLE, clemastine; α-syn, α-synuclein; CTRL, control; WB, western blot; ANOVA, analysis of variance; SEM, standard error of the mean. n = 5 independent experiments.

Article Snippet: Proteins were transferred to PVDF membranes at 30 V for 90 min. Membranes were blocked with 5% non-fat milk in PBST for 1 h and incubated with primary antibodies for 2 h at room temperature: 1:50,000 mouse GAPDH (60004-1-Ig, Proteintech), 1:2,000 mouse p-mTOR (Ser2448) (67778-1-Ig, Proteintech), 1:5,000 mouse t-mTOR (66888-1-Ig, Proteintech), 1:1000 rabbit p-AMPK (Thr183/172) (ab133448, Abcam), 1:1000 mouse p62 (66184-1-Ig, Proteintech), 1:1,000 rabbit LC3-I/II (orb411847, Biorbyt), 1:5,000 mouse P62 (66184-1-Ig, Proteintech), 1:500 mouse α-syn (66412-1-Ig, Proteintech).

Techniques: Phospho-proteomics, Analysis, Control, Comparison, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: Clemastine alleviates Parkinson’s disease pathology by activating the AMPK/mTOR pathway and enhancing autophagic flux

doi: 10.3389/fnagi.2026.1915475

Figure Lengend Snippet: CLE increases LC3 and reduces phosphorylated α-synuclein in dopaminergic neurons (DaNs) of MPTP-treated mice. (A) Immunofluorescence staining for TH (red, marker for DaNs), LC3 (green), and DAPI (blue) in the substantia nigra (SN) of mice from CTRL, MPTP, and MPTP+CLE groups. Scale bar: 2 μm. (B) The quantification of the relative number of LC3 puncta in DaNs. (C) Immunofluorescence staining for TH (red), p-α-syn (Ser129, green), and DAPI (blue) in the SN. Scale bar: 5 μm. (D) The quantification of the relative fluorescence intensity of p-α-syn (Ser129) in DaNs. Data are presented as mean ± SEM (N = 21 per group). Tukey’s multiple comparison test was performed after one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. CLE, clemastine; DaNs, dopaminergic neurons; p-α-syn, phosphorylated α-synuclein (Ser129); SN, substantia nigra; CTRL, control; ANOVA, analysis of variance; SEM, standard error of the mean. n = 21 images per group (7 images from 3 mice per group).

Article Snippet: Proteins were transferred to PVDF membranes at 30 V for 90 min. Membranes were blocked with 5% non-fat milk in PBST for 1 h and incubated with primary antibodies for 2 h at room temperature: 1:50,000 mouse GAPDH (60004-1-Ig, Proteintech), 1:2,000 mouse p-mTOR (Ser2448) (67778-1-Ig, Proteintech), 1:5,000 mouse t-mTOR (66888-1-Ig, Proteintech), 1:1000 rabbit p-AMPK (Thr183/172) (ab133448, Abcam), 1:1000 mouse p62 (66184-1-Ig, Proteintech), 1:1,000 rabbit LC3-I/II (orb411847, Biorbyt), 1:5,000 mouse P62 (66184-1-Ig, Proteintech), 1:500 mouse α-syn (66412-1-Ig, Proteintech).

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, Comparison, Control, Analysis

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 expression is increased in the nigrostriatal system of a monkey model of PD induced by α-syn PFFs injection. (A) Clinical rating score of monkeys after stereotactic injection of 600 μg of α-syn PFFs or an equal volume of normal saline for 4 months. (B) Immunohistochemistry detection of TH in the STR (left) and SN (right). The TH signal intensity in the STR and the number of TH-positive neurons in the SN were lower in the PFF group. The black arrow heads indicate typical TH-positive neurons. Scale bars: 100 μm. (C) Quantitative immunohistochemistry density analysis of TH in the STR. (D) Quantitation of the ratio of TH-positive neurons in the SN in the PFF group compared with the Sham group. (E) Western blot analysis of TH, LRP1, and α-syn expression levels in the monkey STR. (F–H) Densitometric analysis of TH (F), LRP1 (G), and α-syn (H) expression levels in the STR. (I) Western blot analysis of TH, LRP1, and α-syn expression levels in the monkey SN. (J–L) Densitometric analysis of TH (J), LRP1 (K), and α-syn (L) expression levels in the SN. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. Sham group (unpaired t -test). LRP1: Low-density lipoprotein receptor-related protein 1; PD: Parkinson’s disease; PFF: pre-formed fibril; SN: substantia nigra; STR: striatum; TH: tyrosine hydroxylase; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Expressing, Injection, Saline, Immunohistochemistry, Quantitation Assay, Western Blot

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 expression is increased in the nigrostriatal system of a mouse model of PD induced by injection with α-Syn PFFs. (A) Visual gait analysis of walking, gait, and footprint pressure in mice after stereotactic injection with 5 μg of α-Syn PFFs or an equal volume of PBS for 4 weeks ( n = 6). (B) Normal step sequence in mice ( n = 6). (C) Average speed in mice ( n = 6). (D) Tripod support time in mice ( n = 6). (E) Immunohistochemistry staining for TH in the STR. The images in the second row are enlarged images of the areas indicated in black boxes in the upper row. The TH signal intensity in the STR was lower in the PFF group. Scale bars: 200 μm, 40 μm (enlarged). (F) Immunohistochemistry staining for TH in the SN. The images in the second row are enlarged images of the areas indicated in black boxes in the upper row, and the white arrowheads indicate typical TH-positive neurons. There were fewer TH-positive neurons in the SN in the PFF group. Scale bars: 100 μm, 20 μm (enlarged). (G) Quantitative immunohistochemistry density analysis of TH in the STR, compared with the Sham group ( n = 3). (H) Quantitation of the ratio of TH-positive neurons in the SN in the PFF group compared with the Sham group ( n = 3). (I) Western blot analysis of TH, LRP1, and α-Syn expression levels in the mouse STR after injection with 5 μg of α-Syn PFFs or an equal volume of PBS ( n = 3). (J–L) Densitometric analysis of TH (J), LRP1 (K), and α-Syn (L) expression levels in the STR. (M) Western blot analysis of TH, LRP1, and α-Syn expression levels in the mouse SN ( n = 3). (N–P) Densitometric analysis of TH (N), LRP1 (O), and α-Syn (P) expression levels in the SN ( n = 3). Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . Sham group (unpaired t -test). LRP1: Low-density lipoprotein receptor-related protein 1; PBS: phosphate buffered solution; PD: Parkinson’s disease; PFF: pre-formed fibril; SN: substantia nigra; STR: striatum; TH: tyrosine hydroxylase; α-Syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Expressing, Injection, Sequencing, Immunohistochemistry, Staining, Quantitation Assay, Western Blot

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: Exogenous α-syn PFFs upregulate LRP1 expression in PC12 cells. (A) Viability of PC12 cells incubated with different doses (0, 5, 10, 20, or 50 µg/mL) of α-Syn PFFs for 24 hours. ** P < 0.01, *** P < 0.001, vs . 0 µg/mL group. (B) Western blot analysis of LRP1 and α-syn expression levels in PC12 cells. (C, D) Densitometric analysis of LRP1 (C) and α-syn (D) expression levels. (E) Immunofluorescence analysis of LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue) expression. The LRP1 and α-syn signal intensities were stronger in PFF group than in Con group. Scale bars: 40 μm. (F, G) Quantitative immunofluorescence intensity analysis of LRP1 (F) and α-syn (G). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . Con group; # P < 0.05, ## P < 0.01, vs. monomer group. Data are expressed as mean ± SD ( n = 3). Con: Control; DAPI: 4′,6-diamidino-2-phenylindole; PFF: pre-formed fibril; LRP1: low-density lipoprotein receptor-related protein 1; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Expressing, Incubation, Western Blot, Immunofluorescence, Control

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 knockdown rescues the dopaminergic damage induced by exogenous α-syn PFFs. (A, B) Western blot analysis (A) and densitometric analysis (B) of TH expression levels in the STR of mice treated with PFFs. (C, D) Western blot analysis (C) and densitometric analysis (D) of TH expression levels in the SN of mice treated with PFFs. (E) Immunohistochemistry staining for TH in the STR of mice treated with PFFs and LRP1 siRNA. The decrease in relative TH intensity in STR observed in the PFF group was rescued by LRP1 siRNA treatment. The images in the second row are enlarged images of the areas indicated by black boxes in the upper row. Scale bars: 200 μm, 40 μm (enlarged). (F) Quantitative immunohistochemistry density analysis of TH in the STR. (G) Immunohistochemistry analysis of TH in the SN of mice treated with PFFs. The decreased ratio of TH-positive neurons in the SN of the Scramble + PFF group (PFF group) was rescued by LRP1 siRNA treatment. The images in the second row are enlarged images of the areas indicated by black boxes in the upper row, and the white arrowheads indicate typical TH-positive neurons. Scale bars: 100 μm, 20 μm (enlarged). (H) Quantitation of the ratio of TH-positive neurons in the SN compared with the PBS group. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. Scramble + PBS group (PBS group); # P < 0.05, ## P < 0.01, vs. scramble + PFF group (PFF group). LRP1: Low-density lipoprotein receptor-related protein 1; PBS: phosphate buffered solution; PFF: pre-formed fibril; siRNA: small interfering RNA; SN: substantia nigra; STR: striatum; TH: tyrosine hydroxylase; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Knockdown, Western Blot, Expressing, Immunohistochemistry, Staining, Quantitation Assay, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 suppression reduces the transmission of exogenous α-syn in vivo . (A) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue) in the mouse STR after stereotactic injection with α-syn PFFs for 6 weeks with or without LRP1 knockdown. The increase in the relative fluorescence intensity of LRP1 and α-syn in the STR of PFFs-treated mice was rescued by LRP1 siRNA treatment. Scale bars: 100 μm. (B, C) Quantitative immunofluorescence intensity of LRP1 (B) and α-syn (C) in mouse STR. (D) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue) in the SN of mice after stereotactic injection with α-syn PFFs for 6 weeks with or without LRP1 knockdown. The increase in the relative fluorescence intensity of LRP1 and α-syn in the SN of mice treated with PFFs was rescued by LRP1 siRNA treatment. The white arrowheads indicate typical cells exhibiting α-syn and LRP1 expression. Scale bars: 100 μm. (E, F) Quantitative immunofluorescence intensity of LRP1 (E) and α-syn (F) in the mouse SN. (G) Western blot analysis of LRP1 and α-syn expression levels in the mouse STR. (H, I) Densitometric analysis of LRP1 (H) and α-syn (I) expression levels in the mouse STR. (J) Western blot analysis of LRP1 and α-syn expression levels in the mouse SN. (K, L) Densitometric analysis of LRP1 (K) and α-syn (L) expression levels in the mouse SN. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, vs. Scramble + PBS group (PBS group); # P < 0.05, ## P < 0.01, vs . Scramble + PFF group (PFF group). DAPI: 4′,6-Diamidino-2-phenylindole; LRP1: low-density lipoprotein receptor-related protein 1; PBS: phosphate buffered solution; PFF: pre-formed fibril; siRNA: small interfering RNA; SN: substantia nigra; STR: striatum; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Transmission Assay, In Vivo, Immunofluorescence, Staining, Injection, Knockdown, Fluorescence, Expressing, Western Blot, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 mediates the uptake of α-syn PFFs by PC12 cells. (A) Western blot analysis of LRP1 and α-syn expression levels in PC12 cells with or without LRP1 knockdown (10 nM siRNA) after incubation with 10 µg/mL α-syn PFFs for 24 hours. (B, C) Densitometric analysis of LRP1 (B) and α-syn (C) expression. (D) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue). The increase in the relative fluorescence intensity levels of LRP1 and α-syn in the PFF group was rescued by LRP1 siRNA treatment. Scale bars: 40 μm. (E, F) Quantitative immunofluorescence intensity analysis of LRP1 (E) and α-syn (F). Data are expressed as mean ± SD ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . Con group; # P < 0.05, ## P < 0.01, #### P < 0.0001, vs . PFF/– group. Con: Control; DAPI: 4′, 6-diamidino-2-phenylindole; PFF: pre-formed fibril; LRP1: low-density lipoprotein receptor-related protein 1; siRNA: small interfering RNA; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Western Blot, Expressing, Knockdown, Incubation, Immunofluorescence, Staining, Fluorescence, Control, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: Lysine residues in the α-syn N-terminus are vital for LRP1-mediated α-Syn internalization. (A) Heatmap of amino acids in different α-syn domains. (B) Western blot analysis of LRP1 and α-Syn levels in PC12 cells after addition of α-syn PFFs (10 µg/mL) with or without capping of lysine residues for 24 hours. (C, D) Densitometric analysis of LRP1 (C) and α-syn (D) expression levels. (E) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-Syn (red, Alexa Fluor 594), and DAPI (blue). The increase in the relative fluorescence intensity of LRP1 and α-syn in the PFF group was rescued by lysine capping of α-syn. Scale bars: 40 μm. (F, G) Quantitative immunofluorescence intensity analysis of LRP1 (F) and α-syn (G). Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, **** P < 0.0001, vs. Con group; # P < 0.05, ## P < 0.01, vs . PFF group. Con: Control; DAPI: 4′,6-diamidino-2-phenylindole; LRP1: low-density lipoprotein receptor-related protein 1; NHS: sulfo-NHS acetate; PFF: pre-formed fibril; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Control

Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: a Paradigm illustrating the differentiation of ENLs from iPSCs. BMP4, Recombinant human bone morphogenetic protein-4; CHIR, CHIR 99021; RA, Retinoic Acid; SB, SB431542; FGF2, Recombinant Human FGF Basic; AA, ascorbic acid; GDNF, Recombinant Human Glial Derived Neurotrophic Factor; NC, neural crest; ENC, enteric neural crest; ENL, enteric neural lineages. Created in BioRender. Winner, B. (2026) https://BioRender.com/652io1g ( b ) Heatmap showing gene expression of neuronal marker TUBB3 , glial marker GFAP and enteric neuronal markers PHOX2B , ELAVL4 and HOXB3 in iPSC-derived ENLs at days 6, 40 and 70 of differentiation using RT-qPCR. Log 2 fold change was calculated in relation to the Iso group at day 6 of differentiation and averaged for n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations. See also Fig. . Source data are provided as a Source Data file. c Immunocytochemistry characterization of iPSC-derived ENLs at day 70 after start of differentiation. Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right). Scale bar = 50 μm. Figure is representative of two independent experiments. d UMAP plots obtained from scRNAseq analysis of Iso and SNCA 3x ENLs at day 70 after start of differentiation, separated by the clusters identified after annotation. e Compositional analysis plot showing the absence of significantly changed clusters between Iso and SNCA 3x. Data are presented as observed log 2 fold change (center points) +/− 95% confidence intervals (error bars). Confidence intervals were calculated using bootstrap resampling. P -values were determined via permutation testing and adjusted for multiple comparisons using the Benjamini-Hochberg method (adj. p -value). FDR=False Discovery Rate; log2FD=log 2 Fold Difference. n = 3 biological replicates per group. f Dotplot showing the average and percentage of expression of canonical marker genes for each cluster identified in (D). g Ridgeplot comparing the expression of SNCA between each cluster identified in (D). n = 3 biological replicates per group, p-values calculated by unpaired two-tailed Student’s t test.

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Recombinant, Derivative Assay, Gene Expression, Marker, Quantitative RT-PCR, Immunocytochemistry, Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: a Ligand-Receptor pair analysis of scRNAseq data prior to subclustering. Pairs with large mean change and high variance are purple. Pairs failing variance ( ≥ 0.3) or mean ( ≥ 0.3) cut-offs are orange and gray, respectively. b Experimental paradigm depicting iPSC-ENL stimulation with cytokines. Created in BioRender. Winner, B. (2026) https://BioRender.com/a2k344q . c ELISA of total α-syn, normalized by total protein. n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM, * p = 0.0433 by two-way ANOVA with Sidak post-hoc. d Flow cytometry analysis using Live/Dead staining. n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM. e ELISA of aggregated α-syn, normalized by total protein. n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM, * p = 0.0303 by two-way ANOVA with Sidak post-hoc. f Human cytokine array quantification of the supernatants of iPSC-ENLs previously stimulated for 24 h with 100 ng/ml TNF. Data represent the mean of n = 3 independent SNCA 3x and 3 isogenic lines per group, from one differentiation. g Experimental paradigm for the MEA experiments. Created in BioRender. Winner, B. (2026) https://BioRender.com/vwugkr4 . Quantification of the number of spikes ( h ), and active electrodes ( i ), n =wells of a CytoView MEA 48-well plate, representative of n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM, * p = 0.046, ** p = 0.0031 by two-way ANOVA with Sidak post-hoc. j Flow cytometry analysis of the percentage and total of α-syn + (2A7 antibody) enteric neurons (CD56 + CD24 high ) and enteric glia (CD56 + CD24 low ). n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM, p = 0.0419 (% α-syn + neurons); p = 0.0294 (# α-syn + neurons); p = 0.0478 (% α-syn + glia); p = 0.0123 (# α-syn + glia) by two-way ANOVA with Sidak post-hoc. k Flow cytometry analysis of the percentage and total enteric neurons (CD56 + CD24 high ) and enteric glia (CD56 + CD24 low ). n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM. Source data are provided as a Source Data file in c – f , h , I , j , k . Basal: DPBS + 0.1% BSA.

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining

Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: a Paradigm describing the proximity ligation assay (PLA) and measurement of mitochondrial ROS experiments. Created in BioRender. Winner, B. (2026) https://BioRender.com/vb5oi60 . b Assessment of PLA results using immunocytochemistry. Panels are separated in basal and TNF stimulated for both Iso and SNCA 3x groups. Panels show the nuclear marker DAPI (blue), the mitochondrial marker TOM20 (gray), α-syn (green) and PLA dots for TOM20-α-syn (red). Scale bar = 50 μm. Basal refers to cells treated with vehicle used to dilute the TNF (DPBS + 0.1% BSA). c Quantification of the PLA dots in the cytoplasm. n=number of total individual cells analyzed per condition indicated in the figure, from 3 independent SNCA 3x and 3 isogenic lines per group, from one differentiation, with mean ± SEM, **** p < 0.0001, by one-way ANOVA with Tukey’s post-hoc. Basal refers to cells treated with vehicle used to dilute the TNF (DPBS + 0.1% BSA). Data were normalized to the number of nuclei per image. Source data are provided as a Source Data file. Concatenated t-SNE plots from flow cytometry data of all lines highlighting Mitosox high populations across genotypes and conditions and quantification of the percentage and total mitosox high cells in enteric neurons (CD56 + CD24 high ) ( d ) and in enteric glia (CD56 + CD24 low ) ( e ). n = 3 independent SNCA 3x and 3 isogenic lines per group, from two independent differentiations, mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Proximity Ligation Assay, Immunocytochemistry, Marker, Flow Cytometry

Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: a Paradigm of meta-analysis of independent UC patient cohorts, responders or non-responders to infliximab therapy and controls ( GSE16879 , GSE12251 , GSE73661 ). Created in BioRender. Winner, B. (2026) https://BioRender.com/jkisazh . b GOT1 expression (log2 normalized) across datasets. Analyzed by Student’s t -test or one-way ANOVA (Tukey’s post-hoc). GSE73661 : Control vs. UC NR before, p = 0.0002; Control vs. UC R before, p = 0.035; UC R before vs. UC R after, p = 0.0421. GSE16879 : Control vs. UC NR before, p = 0.0007. GSE12251 : p = 0.0158. c SNCA expression (log2 normalized) across datasets. Analyzed by t-test or ANOVA. GSE73661 : Control vs. UC NR before, p = 0.0237; UC NR after vs. UC R after, p = 0.0112. d Bioinformatic analysis paradigm of UC cohorts (inflamed/non-inflamed) using bulk RNA-seq (IBDome, https://ibdome.org/ ) and scRNAseq (scIBD, http://scibd.cn/ ). Created in BioRender. Winner, B. (2026) https://BioRender.com/h6tkzor . e Expression of MAS genes ( GOT1 , GOT2 , MDH1 , MDH2 ) in inflamed vs. non-inflamed UC tissue (IBDome, n = 66 inflamed, n = 28 non-inflamed). TPM normalized, log10 scale. Two-sided Wilcoxon-Mann-Whitney test. Error bars: 95% CI. Exact p-values are indicated in the figure. f SNCA expression in inflamed vs. non-inflamed UC tissue (IBDome, n = 66 inflamed, n = 28 non-inflamed). TPM normalized, log10 scale. Two-sided Wilcoxon–Mann–Whitney test. Error bars: 95% CI. g Correlation analysis between GOT1 and SNCA in inflamed and non-inflamed tissue (IBDome, n = 66 inflamed, n = 28 non-inflamed). Pearson test. The shaded area denotes the 95% confidence interval of the slope of the linear model. h UMAP plot generated by integrating the 12 scRNAseq studies from the scIBD platform, highlighting the neural cluster in red. i Mean expression value of the MAS enzymes GOT1 , GOT2 , MDH1 and MDH2 in healthy, UC non-inflamed and UC inflamed tissue. Data came from the scIBD platform. j Immunocytochemistry of healthy controls and UC patient gut tissue: β-tubulin III (orange), total α-syn (red), AATC (green), DAPI (blue). Scale bar: 50 μm. k Quantification of the immunocytochemistry shown is (J) for n = 3 controls and n = 3 UC patients, mean ± SEM. Integrated density was calculated using FIJI and normalized to the total cell count per image. Data analyzed by unpaired two-tailed t -tests. AATC, * p = 0.04; α-syn, * p = 0.01. Source data are provided as a Source Data file in b , c , e , f , g , k .

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Expressing, Control, RNA Sequencing, MANN-WHITNEY, Generated, Immunocytochemistry, Cell Characterization, Two Tailed Test

Journal: Nature Communications

Article Title: TNF alpha unmasks enteric malate aspartate shuttle dysfunction bridging Parkinson disease and intestinal inflammation

doi: 10.1038/s41467-026-71317-y

Figure Lengend Snippet: Graphical summary of findings. SNCA 3x drives inflammatory-metabolic vulnerability in the ENS. Our multi-omics and functional approach using iPSC-derived ENLs revealed that α-syn accumulation induces basal mitochondrial and synaptic dysfunction, altering cell composition and transcriptional profiles in both enteric neurons and glia. Challenging these cells with the proinflammatory cytokine TNF acts as a second-hit, unmasking selective vulnerability in SNCA 3x ENLs, marked by enhanced α-syn-mitochondria interactions and a corresponding surge in oxidative stress-associated populations. This functional collapse is mechanistically driven by a disruption of the malate-aspartate shuttle and the TCA cycle, forcing cells to become critically reliant on glutamine oxidation for energy. We demonstrate that the glutamate metabolism modulator Chicago Sky Blue 6B effectively restores mitochondrial function, reverses the TNF-driven glutamine dependency, and enhances metabolic flexibility, suggesting a targeted therapeutic strategy for α-syn-driven enteric pathology. Finally, we validate this core mechanism by showing that MAS suppression, linked to α-syn accumulation, is a conserved signature in inflamed human gut tissue. Created in BioRender. Winner, B. (2026) https://BioRender.com/d91886y .

Article Snippet: Panels show the glial marker GFAP (red), the enteric neuronal marker HuC/D (green), α-syn (green, 2A7 antibody, Novus, Cat#NBP1-05194) and DAPI (blue) for cell nuclei staining for both Iso (left) and SNCA 3x (right).

Techniques: Biomarker Discovery, Functional Assay, Derivative Assay, Disruption

Journal: bioRxiv

Article Title: A new therapeutic approach for Parkinson’s disease: dual targeting of α-Synuclein aggregation and microglial function by the novel immunomodulator 3-Monothiopomalidomide

doi: 10.64898/2026.03.26.714051

Figure Lengend Snippet: ( A ) ThT fluorescence intensity monitored during α-Syn (100 μM) aggregation at 37 °C, in PBS at pH 7.4 and under orbital shaking (200 rpm). Kinetic traces for isolated α-Syn (black) and in co-incubation with POM (yellow) and 3MP (orange) are reported. All conditions were tested in triplicate. The intrinsic fluorescence of 3MP was found to be negligible compared to the intensity of ThT fluorescence. ( B ) t½ values of the kinetic curves. ( C ) Plateau values of the kinetic curves. One-way ANOVA followed by Dunnett’s post-hoc test was performed against protein alone (control). ns = not significant, * p < 0.05, *** p < 0.001. Shapiro–Wilk and Bartlett’s tests confirmed normality and homogeneity of variance. ( D ) Transmission electron microscopy (TEM) images of samples post 120h of incubations. The images were taken at both 100 nm and 200 nm magnifications. Samples were prepared using negative staining with 2% phosphotungstic acid (PTA) on carbon-coated 200 mesh copper grids. ( E ) Representative images of ThS staining on SH-SY5Y cells challenged with α-Syn pre-formed fibrils and treated with POM and 3MP (magnification 63x; scale bar: 20µm). ( F ) Quantification of ThS + signal expressed as ThS□ volume per cell (µm³/cell) in SH-SY5Y cells challenged with α-Syn pre-formed fibrils and treated with POM or 3MP. Data are shown as mean ± SEM from three independent biological replicates. One-way ANOVA followed Tuckey’s post-hoc test.

Article Snippet: Cells were pre-treated for 1 hour with 60 μM of POM or 3MP, followed by the addition of 1 μM of α-Syn pre-formed fibrils (PFFs; StressMarq Biosciences Inc., #SPR-317) to the culture medium.

Techniques: Fluorescence, Isolation, Incubation, Control, Transmission Assay, Electron Microscopy, Negative Staining, Staining

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 shows high labeling accuracy for α-syn aggregates in vitro, compared to phosphorylated (pS129) α-syn antibody which was used as the gold standard reference staining for α-syn aggregates. (a) Structure of peptide-based probe P1 ; (b) Setup of primary hippocampal neuronal culture and induction of α-syn aggregation upon addition of PFFs; (c) Confocal microscopy of primary neuronal cells post 7 days of PFF addition. Nuclei are colored blue, total α-syn green, pS129 α-syn orange, and probe P1 in red. Scale bar represents 200 μm; (d) Probe P1 shows colocalized staining with the PS129 α-syn antibody staining. The probe labels most of the same focal locations, but with slightly larger surrounding area than the PS129 α-syn antibody hitsthis is still regarded as a positive colocalization. True positives (indicated by green arrows) are stained by both P1 and the pS129 α-syn antibody. False negatives (indicated by pink arrows) are not stained by P1 but stained by the pS129 α-syn antibody. Scale bar represents 100 μm; (e) Graph showing average positive predictive value (true positives/probe positives) of 87.4% and miss rate (false negatives/antibody positives) of 8.7%. Values on graph are given as the mean ± SEM calculated over 10 wells, with 9 fields of view each.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Labeling, In Vitro, Staining, Confocal Microscopy

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 exhibits preferential binding to α-syn fibrils over α-syn monomers. (a) Fluorescent excitation and (b) fluorescent emission spectra of P1 . 2 μM probe P1 was incubated with 10 μM of various α-syn species, including monomers, A* oligomers, B* oligomers, and PFFs. Concentrations of all α-syn species are expressed at their equivalent monomer concentration. Fluorescence intensity of P1 increases significantly upon interaction with PFFs (7.6× and 16× for excitation and emission respectively); (c) Fluorescence titration curve of P1 (0.5 μM) to PFFs, showing micromolar K D ; (d) Native-PAGE images of P1 with various α-syn species, showing that P1 preferentially binds to B* oligomers and PFFs, over α-syn monomers and A* oligomers. 2 μg of P1 was incubated with 2 μg of α-syn monomers and various α-syn aggregate species and run under native PAGE conditions. In-gel fluorescence was first visualized in the 647 channel, and then the gel was subsequently imaged in the brightfield channel after staining with Coomassie blue. Red bands correspond to P1 bound to the B* oligomers and PFFs; (e) Dot-blot images of P1 (0.5 μM) with various human α-syn species, showing concentration-dependent labeling of kinetically stable oligomers and PFFs.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Binding Assay, Incubation, Concentration Assay, Fluorescence, Titration, Clear Native PAGE, Staining, Dot Blot, Labeling

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Probe P1 shows specific staining of α-syn aggregates in tissues from PFF-injected mouse models. (a) Models used for ex vivo tissue labeling: PFF-injected mouse (C57BL6), where PFF was injected directly into the brain or duodenum, and transgenic mouse models overexpressing human α-syn; (b) Confocal microscopy of brain tissues after 30 days of PFF injection showing high levels of α-syn aggregation in the hippocampal and cortex regions, labeled specifically by both pS129 α-syn antibody and P1 ; (c) Confocal microscopy of duodenum tissues after 75 days of PFF injection showing low levels of α-syn aggregation in the mucosa region indicated by the white arrows. Images of tissues from control PBS-injected mice are shown in Figure S5 for comparison; (d) Confocal microscopy of brain tissue (substantia nigra) of a healthy control and of a PD patient (post-mortem), with pS129 α-syn antibody staining and P1 staining observed in the substantia nigra of the PD patient; (e) Confocal microscopy showing Lewy bodies (LB) and Lewy neurites (LN) highlighted by pS129 α-syn antibody staining in the substantia nigra of a PD patient. Higher magnification images of LB and LN show specific labeling by P1 that corresponds to the antibody staining. Nuclei in blue, α-syn in green, pS129 α-syn in orange and probe P1 in red.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Staining, Injection, Ex Vivo, Labeling, Transgenic Assay, Confocal Microscopy, Control, Comparison

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Specific α-syn antibody and probe P1 labeling of entire GI tract (esophagus, stomach, duodenum, jejunum, ileum, and colon) in transgenic mice (JAX004479). (a) Aggregated α-syn is observed in the mucosa layers and stained by both pS129 α-syn antibody and P1 (annotated with white arrows). Tissue layers are indicated by black arrows: muscularis externa (ME), submucosa (SM), and mucosa (M). Nuclei are in blue, α-syn in green, pS129 α-syn in orange, and probe in red. Scale bar represents 200 μm; (b) Total expression areas of α-syn aggregates labeled by pS129 α-syn antibody and P1 were quantified, with the highest expressions observed in the esophagus and colon; (c) High degree of colocalization (average of 0.91) was observed between pS129 α-syn antibody and probe P1 channels across all the GI tract tissues, determined by the Costes method. Manders colocalization and correlation coefficients are shown in Figure S7a ; (d) Probe positive predictive values were consistent across the respective GI tract tissues. Values on graphs are given as the mean ± SEM ( n = 3 per group); (e) P1 labeling is observed in the enteric neurons in colon tissue, marked by neuronal marker TUBB3 (green).

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Labeling, Transgenic Assay, Staining, Expressing, Marker

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Increased accumulation of α-syn aggregates across GI tissues in older transgenic mice (JAX010799). (a) Western blot showing abundance of total α-syn in brain, esophagus, duodenum, and colon of young (5-month) and old (15-month) transgenic mice (JAX010799), with higher molecular weight α-syn species observed in the older mice (indicated by red arrows); (b) Graph showing intensity of bands corresponding to α-syn, normalized to beta-actin, quantified from blots in Figure S7 . Older mice showed increased intensities of α-syn bands; Confocal images showing increased staining of aggregated α-syn in (c) esophagus and (d) colon of older transgenic mice. Tissue layers as indicated by black arrows: muscularis externa (ME), submucosa (SM), mucosa (M). Scale bar represents 200 μm; (e) Graphs showing degree of α-syn aggregation determined by pS129 α-syn antibody labeling and P1 labeling, with generally higher degrees of α-syn aggregation observed across all GI tissues in older mice. Values on graphs are given as the mean ± SEM ( n = 3 per group). Ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, determined by two-way ANOVA test.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Transgenic Assay, Western Blot, Molecular Weight, Staining, Antibody Labeling, Labeling

Journal: ACS Chemical Neuroscience

Article Title: Fluorescence Detection of Alpha-Synuclein Aggregates in the Gut Using a Peptide Probe

doi: 10.1021/acschemneuro.6c00069

Figure Lengend Snippet: Longitudinal view of unrolled colon tissue of transgenic mouse showing differing levels of α-syn aggregation across tissue layers (mucosa, submucosa and muscularis externa). (a) Confocal microscopy of selected section of colon tissue from transgenic mouse, with the different tissue layers marked by white dashed lines. α-Syn aggregates on the mucosa surface are specifically labeled by pS129 α-syn antibody (orange) and P1 (red). Full view of section is shown in Figure S8 ; (b) Graph showing degree of α-syn aggregation (pS129 α-syn antibody or P1 positives, divided by α-syn positives) in the mucosa, submucosa, and muscularis externa regions. Mucosa shows lower but detectable levels of P1 staining compared to the other tissue layers; (c) Confocal microscopy of colon tissue from a transgenic mouse, flushed with P1 (15 μg/mL), followed by fixation and immunofluorescence staining with pS129 α-syn antibody (orange) and aggregated α-syn antibody, clone 5G4 (green). P1 labels α-syn aggregates in the surface mucosal layer.

Article Snippet: 15 μL of 2 mg/mL α-syn PFF (StressMarq SPR-324) were sonicated in an ultrasonic bath sonicator for 2.5 h at the following conditions: sweep, 37 Hz frequency, 60% power.

Techniques: Transgenic Assay, Confocal Microscopy, Labeling, Staining, Immunofluorescence