p301s mutation  (ATCC)

Journal: Autophagy

Article Title: Impaired MAPT/tau-secretory lysosomes are linked to cognitive vulnerability in Alzheimer patients

doi: 10.1080/15548627.2025.2552905

Figure Lengend Snippet: Presence of lysosomal-resistant MAPT/tau fibrils in AD brains. (A) lysosomes isolated from HEK 293 cells were incubated with recombinant monomeric P301S MAPT/tau for varying time periods. Western blotting probed with tau-5 antibodies shows a decrease in MAPT/tau levels with increasing incubation time. (B) lysosomes isolated from HEK 293 cells were incubated with cell lysates prepared from normal and AD postmortem brain tissues. Western blotting probed with tau-5 antibodies reveals that MAPT/tau from AD brain lysates is more resistant to lysosomal degradation compared to MAPT/tau from normal brain lysates. (C) lysosomes isolated from normal and AD brains were probed with tau-5 and 3 R antibodies via western blotting. The results demonstrate the presence of undigested MAPT/tau within lysosomes from AD brains. (D) Coimmunoprecipitation was performed from AD brain lysates using LAMP1 antibodies, followed by probing with tau-5 and LAMP1 antibodies for western blotting. The results show that MAPT/tau coimmunoprecipitates with LAMP1, indicating their association. (E, F) Representative immunohistochemistry (IHC) images show pTau (AT8) colocalized with LAMP1 in human AD brain tissues. Arrows indicate the colocalized AT8/LAMP1 puncta. Scale bar: 5 μm (E). Quantitative analysis reveals a significant reduction in AT8 − LAMP1 + (Mapt/tau-free lysosomes) in AD compared to normal ( n = 3) (F). (G) a negative staining transmission electron microscopy (TEM) image shows the presence of MAPT/tau fibrils within lysosomes isolated from AD brains. Arrows indicate MAPT/tau fibrils. Scale bar: 100 nm. (H) lysosomal MAPT/tau derived from AD brains induced MAPT/tau aggregates in MAPT/tau FRET biosensor cells. Scale bar: 5 μm. (I, J) western blotting of lysosomes isolated from CDR-2 and CDR-5 groups probed with the MAPT/tau (3 R) antibody shows an increased level of MAPT/tau in lysosomes from CDR-5 compared to the CDR-2 ( n = 8). Relative MAPT/tau expression was quantified by measuring band intensities and normalizing them to corresponding total protein. (K) Sex-based analysis showed no significant difference in lysosomal MAPT/tau levels between male and female cases within the CDR-2 group. However, in the CDR-5 group, female exhibited higher MAPT/tau levels than male ( n = 4). Significance levels: *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: MAPT/tau FRET biosensor cells (HEK-293) cells that express MAPT/tau repeat domain having P301S mutation fused with CFP and YFP (ATCC, CRL‐3275) were cultured in 24 well plates at 5% CO 2 , 37°C.

Techniques: Isolation, Incubation, Recombinant, Western Blot, Immunohistochemistry, Negative Staining, Transmission Assay, Electron Microscopy, Derivative Assay, Expressing