Journal: bioRxiv

Article Title: Non-neuronal, TGF-β–driven extracellular matrix restructuring promotes neurodegeneration in a PSP-Richardson syndrome model

doi: 10.1101/2025.08.17.670724

Figure Lengend Snippet: a, b, Immunoblot quantification of phosphorylated AKT (p-AKT^S473) and ERK1/2 (p-ERK1/2^T202/Y204) in MOs from HC and PSP-RS donors shows increased activation of both pathways in PSP-RS (mean ± s.e.m.; n = 3; P = 0.0269 for p-AKT, P = 0.0203 for p-ERK1/2; unpaired t-test with Welch’s correction). c, Expression of the PP2A phosphatase is significantly reduced in PSP-RS MOs (P = 0.0179). d, Treatment of PSP-RS MOs with the PI3K inhibitor LY294002 reduces p-AKT levels and diminishes phosphorylation of tau at S396 and T181 (P = 0.0081, P = 0.0051, and P = 0.0014, respectively). e, Inhibition of ERK1/2 signaling with PD0325901 leads to decreased p-ERK1/2 and reduced phospho-tau at S396 and T181 in PSP-RS MOs (P = 0.0204, P = 0.0103, and P = 0.0146, respectively). f, Pharmacological blockade of TGF-β signaling with SB431542 reduces p-AKT and p-ERK1/2, as well as phospho-tau at S396 and T181 in PSP-RS cultures (P = 0.0255, P = 0.0314, P = 0.0059, and P = 0.0167, respectively). Band intensities are normalized to total protein or histone H3 (H3) as loading controls. Uncropped western blot gels and statistics are available in Additional File 1-2.

Article Snippet: In PSP-RS organoids, TGFβ pathway inhibition was achieved using the selective ALK5 inhibitor SB431542 (10 μM; Miltenyi Biotec).

Techniques: Western Blot, Activation Assay, Expressing, Phospho-proteomics, Inhibition

Journal: bioRxiv

Article Title: Non-neuronal, TGF-β–driven extracellular matrix restructuring promotes neurodegeneration in a PSP-Richardson syndrome model

doi: 10.1101/2025.08.17.670724

Figure Lengend Snippet: a, Representative images of F-actin staining (phalloidin, red) in MOs primary cultures from healthy controls (HC) and PSP-RS patients. DNA is counterstained in blue (DAPI). Insets show higher magnification of boxed areas. Scale bar, 50 µm. b, Quantification of mean fluorescence intensity (MFI) for F-actin reveals significantly increased actin polymerization in PSP-RS (mean ± s.e.m.; n = 8 images; *** P = 0.0002, unpaired t -test with Welch’s correction). c, Western blot showing reduced expression of actin-capping protein CapZ in PSP-RS compared to HC. d, Western blot of acetylated α-tubulin (Lys40) shows decreased levels in PSP-RS MOs. Uncropped western blot gels are available in Source Data e, Representative images and quantification of RhoA-GTP levels (green) show increased active RhoA in PSP-RS cells (mean ± s.e.m.; n = 60 cells HC, 61 cells PSP; **** P < 0.0001, Mann–Whitney test). DNA, DAPI (blue). Scale bar, 25 µm. f, Treatment of PSP-RS MOs with ROCK inhibitor Y-27632 reduces F-actin intensity (mean ± s.e.m.; n = 8 images HC, 7 images PSP; P = 0.0490, unpaired t -test with Welch’s correction). Scale bar, 50 µm. g, Exogenous TGF-β treatment of HC MOs increases F-actin intensity (mean ± s.e.m.; n = 23 ROIs HC, 24 ROIs HC+TGFβ; ** P = 0.0052, unpaired t -test with Welch’s correction). Scale bar, 50 µm. h, TGF-β I receptor inhibitor SB431542 reduces F-actin intensity in PSP-RS cultures (mean ± s.e.m.; n = 23 ROIs PSP, 26 ROIs PSP+SB; **** P < 0.0001, unpaired t -test with Welch’s correction). Scale bar, 50 µm. Statistics are available in Additional File 2.

Article Snippet: In PSP-RS organoids, TGFβ pathway inhibition was achieved using the selective ALK5 inhibitor SB431542 (10 μM; Miltenyi Biotec).

Techniques: Staining, Fluorescence, Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Non-neuronal, TGF-β–driven extracellular matrix restructuring promotes neurodegeneration in a PSP-Richardson syndrome model

doi: 10.1101/2025.08.17.670724

Figure Lengend Snippet: a, Representative confocal images of MOs from HC and PSP-RS donors stained for synaptophysin (SYP, red), PSD95 (green), and DNA (DAPI, blue) show reduced pre- and post-synaptic puncta in PSP-RS. Scale bar, 10 μm. b, Quantification of synaptic markers reveals decreased fluorescence intensity for SYP (P = 0.0273) and PSD95 (**** P < 0.0001), along with fewer synaptic elements per nucleus (P = 0.0362) in PSP-RS (mean ± s.e.m.). c, Expression of the dendritic spine marker drebrin is significantly reduced in PSP-RS MOs (mean ± s.e.m.; n = 17 images; P = 0.0210). Scale bar, 50 μm. d, Representative images of DA neurons stained for MAP2 (red) reveal diminished neurite complexity in PSP-RS cultures. Scale bar, 25 μm. e, Quantification shows significantly shorter dendritic branch length (*** P = 0.0007) and fewer branches per neuron (** P = 0.0074) in PSP-RS DA neurons (mean ± s.e.m.; n = 14–15 neurons). f, g, Treatment of PSP-RS neurons with the TGF-β receptor inhibitor SB431542 rescues dendritic complexity, increasing both branch length (**** P < 0.0001) and number of branches per neuron (*** P = 0.0005; n = 35 neurons). Scale bar, 25 μm. h, Exogenous TGF-β treatment of HC MOs reduces SYP and PSD95 puncta, mimicking the PSP-RS synaptic phenotype. Scale bar, 25 μm. i, Quantification confirms significant reductions in SYP (**** P < 0.0001), PSD95 (**** P < 0.0001), and synaptic elements per nucleus (*** P = 0.0005) following TGF-β treatment (mean ± s.e.m.; n = 30–32 regions of interest). Statistics are available in Additional File 2.

Article Snippet: In PSP-RS organoids, TGFβ pathway inhibition was achieved using the selective ALK5 inhibitor SB431542 (10 μM; Miltenyi Biotec).

Techniques: Staining, Fluorescence, Expressing, Marker