Journal: International Journal of Molecular Sciences

Article Title: Mutant Tau (P301L) Enhances Global Protein Translation in Differentiated SH-SY5Y Cells by Upregulating mTOR Signalling

doi: 10.3390/ijms27010455

Figure Lengend Snippet: Tau (P301L) enhances rates of global protein synthesis. The rate of protein synthesis was measured via the SUnSET protocol. This evaluation was performed in both RA-differentiated ( A ) and proliferative SH-SY5Y cells ( B ), comparing SH-Tau with SH-wt. GAPDH was quantified to normalise samples. Puromycin/GAPDH levels were graphed for respective cell culture types, normalising against controls (SH-wt) ( C , D ). Statistical evaluation of results was performed by t -test, two-tailed, unpaired, and homoscedastic. * p < 0.05. Six biological replicates were employed for differentiated SH-SY5Y cells, and three replicates for proliferative ones. Error bars indicate s.e.m.

Article Snippet: The human neuroblastoma cell line SH-SY5Y (CRL-2266TM, ATCC, Manassas, VA, USA) and the SH-SY5Y cell line overexpressing the TAU P301L mutation ( P30722 , Innoprot, Derio, Bizkaia, Spain) were used for the experiments.

Techniques: Cell Culture, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Mutant Tau (P301L) Enhances Global Protein Translation in Differentiated SH-SY5Y Cells by Upregulating mTOR Signalling

doi: 10.3390/ijms27010455

Figure Lengend Snippet: Tau (P301L) increases mTOR signalling. Western blot analyses of p-mTOR, mTOR, p-S6, and S6 proteins between differentiated SH-Tau and SH-wt cells ( A ). GAPDH was used to normalise samples relative to mTOR and S6 levels. P-mTOR and p-S6 were compared to their respective total protein levels (i.e., mTOR and S6, respectively). Protein levels were graphed, normalising against controls (SH-wt) ( B ). Statistical evaluation of results was performed by t -test, two-tailed, unpaired, and homoscedastic. * p < 0.05, ** p < 0.01, *** p < 0.001. Three biological replicates were employed for each condition. Error bars indicate s.e.m.

Article Snippet: The human neuroblastoma cell line SH-SY5Y (CRL-2266TM, ATCC, Manassas, VA, USA) and the SH-SY5Y cell line overexpressing the TAU P301L mutation ( P30722 , Innoprot, Derio, Bizkaia, Spain) were used for the experiments.

Techniques: Western Blot, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Mutant Tau (P301L) Enhances Global Protein Translation in Differentiated SH-SY5Y Cells by Upregulating mTOR Signalling

doi: 10.3390/ijms27010455

Figure Lengend Snippet: Tau (P301L) enhances nascent protein synthesis by activating mTOR signalling. S6 and p-S6 protein levels were quantified via Western blotting among four sample conditions: SH-wt rapa−, SH-wt rapa+, SH-Tau rapa−, and SH-Tau rapa+ ( A , up). GAPDH was used to normalise samples relative to S6 levels. In contrast, p-S6 was compared to S6 protein levels and graphed ( A , bottom). Western blot analyses of puromycin-labelled peptides under the same conditions ( B , left). Puromycin levels were normalised against GAPDH protein and graphed ( B , right). Throughout the figure, protein levels were graphed, normalising against controls (SH-wt rapa−). Statistical evaluation of results was performed by t -test, two-tailed, unpaired, and homoscedastic. * p < 0.05, ** p < 0.01. Three biological replicates were employed for each condition. Error bars indicate s.e.m.

Article Snippet: The human neuroblastoma cell line SH-SY5Y (CRL-2266TM, ATCC, Manassas, VA, USA) and the SH-SY5Y cell line overexpressing the TAU P301L mutation ( P30722 , Innoprot, Derio, Bizkaia, Spain) were used for the experiments.

Techniques: Western Blot, Two Tailed Test

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