Fig 2: Phenotypic changes of VSMCs under AD-like conditions in vitro(A) Immunofluorescence staining of primary human VSMCs stimulated with PDGF-BB, TauP301L, LPS, or DMSO for 72 hrs. Top image shows morphological changes in the cytoskeleton (F-actin, green), scale bars: 50 µm. Lower image shows increased Tau (red) and MMP9 (green) expression in VSMCs under AD-like conditions. Scale bars: 30 µm.(B) Heatmap of mRNA expression of VSMCs under AD-like conditions versus control group (DMSO). Left image shows classic VSMC-contractile markers. Middle image shows VSMC synthetic makers, and right image shows inflammatory markers. Red indicates upregulated genes, white indicates no significant change, and blue indicates downregulated genes. *p < 0.05, **p < 0.01, ***p < 0.001 level of significance in genes upregulated as compared to control (DMSO). Respectively, compared to the corresponding control, it is determined by one-way analysis of variance (ANOVA).(C) Flow cytometry analysis of CD68 and IL-6R expression in VSMCs under AD-like conditions.(D) Would healing assay of co-cultured human VSMC and microglia cells. Microglia were labeled with CellTracker Deep Red Dye and DAPI nuclear stain. VSMCs were labeled with CellTracker Green Dye. The migratory activity of VSMCs and microglia were observed at 0, 12, 24, and 48 hr and quantified as the percentage wound healing area occupied by cells. Scale bars: 30 µm. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to controls. Respectively, compared to the corresponding control, it is determined by one-sample t test.

Fig 3: Heroin leads to FYN-mediated hyperphosphorylation of Tau protein.a Immunocytochemistry analysis of vehicle and morphine-treated primary cortical-striatal co-cultured neurons revealed increased phosphorylation of Tyrosine-18 of Tau in morphine-treated cells (n = 3 biological replicates/group, Student’s t-test, t4 = 4.374, p = 0.0119). ×40 magnification, scale bars represent 33 µm. b, c Western blots from the caudate-putamen of heroin self-administering rats showed Tau hyperphosphorylation in vivo both 1 h (B; n = 5/group, Student’s t-test, t8 = 2.359, p = 0.0461) and 24 h (c; n = 6/group, Student’s t-test, t10 = 2.503, p = 0.0313) following the last self-administration session. Data are represented as mean ± SEM. DAPI 4,6-diamidino-2-phenylindole. Source data are provided as a Source Data file.

Fig 4: The FTLD P301L mutant Tau promotes the lateral trapping of Fyn-mEos2 in neurons and HEK-293T cells, altering the downstream ERK/S6 signalling.A Representative epifluorescence image of GFP co-expressed in hippocampal dendrites with Fyn-mEos2. The white box outline is shown at a higher magnification below to display the intensity map of Fyn-mEos2. B Representative epifluorescence image of Tau-P301L-GFP co-expressed in hippocampal dendrites with Fyn-mEos2. The white box outline is shown at a higher magnification below to display the intensity map of Fyn-mEos2. C Mobility of Fyn-mEos2 co-expressed with GFP or with Tau-P301L-GFP in hippocampal neurons indicated as the MSD (µm2) curves over time (0.14 s). D Corresponding AUC (µm2 s) of the graphs in C. E, F Representative diffusion coefficient maps of Fyn-mEos2 co-expressed with E GFP, or F Tau-P301L-GFP in HEK-293T cells. White boxed outlines are shown magnified on the right in (i). Note that hotter colours within diffusion coefficient maps designate regions of lower mobility. G Mobility of Fyn-mEos2 co-expressed with GFP or with Tau-P301L-GFP in HEK-293T cells indicated as the MSD (µm2) curves over time (0.14 s). H Corresponding AUC (µm2 s) of the graphs in G. I Quantification of the % of clustered Fyn-mEos2 trajectories upon co-expression with GFP or with Tau-p301L-GFP. J Quantification of the density of Fyn-mEos2 clusters (clusters/µm2) upon co-expression with GFP or with Tau-p301L-GFP. K Western blot of HEK-293T cells transfected with either Fyn-myc and an empty vector, Tau-P301L-V5 and an empty vector, or Fyn-myc and Tau-P301L-V5. L Analysis of Tau phosphorylation at the Y18 epitope (pTau/Tau), M ERK1/2 activity (pERK/ERK) and N S6 activity (pS6/S6) measured using the relative intensity of the corresponding western blot bands. Error bars are standard errors of the mean (SEM). Mean ± SEM values in C, D were obtained from hippocampal neurons transfected with Fyn-mEos2 and GFP (N = 15) or Tau-P301L-GFP (N = 15). Mean ± SEM values in G–J were obtained from HEK-293T cells transfected with Fyn-mEos2 and GFP (N = 10 in G, H and N = 13 in I, J) or Tau-P301L-GFP (N = 9 in G, H and N = 22 in I, J). Mean ± SEM values in L–N were obtained from N = 3. Statistical comparisons in D, H, I and J were performed using unpaired Welch’s t-test. Statistical comparisons in L–N were performed using a one-way ANOVA and Tukey’s test for multiple comparisons between groups. The specific adjusted p values accounting for multiple comparisons are reported when the data are considered significantly different (p < 0.05).

Fig 5: Dysfunctional VSMCs induce Tau hyperphosphorylation and an in silico analysis of mutant Tau proteins(A) Representative western blots shows levels of total Tau (t-Tau) and multiple phosphorylate Tau residues (p-tau-Y18, p-tau-S262, p-tau-T205) from total lysates of VSMCs under AD-like conditions. CFL-1 (Coflin-1, a proliferative marker of VSMC). VCL was used as loading internal control.(B and C) In silico analysis of the WT and mutant TauP301L proteins.(D) Plot of distance (nm) vs time of the Y18 residue and R2-R3 domains after 500 ns of molecular dynamics simulations (MDSs) shows a significant reduction between the Y18 residue and the L301 mutant residue.