Journal: iScience

Article Title: TXNRD1-driven enteric glial remodeling promotes neuronal dysfunction in Hirschsprung disease

doi: 10.1016/j.isci.2026.117636

Figure Lengend Snippet:

Article Snippet: Mouse anti-Synaptophysin (SYN) , Santa Cruz Biotechnology , Cat# sc-365488.

Techniques: Model Organisms, Recombinant, CCK-8 Assay, Apoptosis Assay, ROS Assay, Calcium Assay, Enzyme-linked Immunosorbent Assay, Generated, Software, Genotyping

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