Table 1.
shRNA sequence data for human PINCH1 and 2.
Table 2.
PINCH deletion mutants and sequences.
Figure 1.
Exposure of neurons to TNF-α results in increased levels of PINCH, hp-Tau and CHIP.
Representative Western blot of A) neurons exposed to TNF-α; B) graphic representation of fold change of PINCH1, hp-Tau, total Tau and CHIP levels in TNF-α treated neurons over control. Results are from 4 separate experiments and are expressed as fold change over control. * p<0.005, **p<0.001 by one-way ANOVA with Tukey-Kramer post-hoc analyses. Grb-2 was used as the loading control. C) Fold change in hp-Tau/Total Tau * p = 0.0293 by one-tailed paired T-test.
Figure 2.
Exposure of neurons to supernatant from HIV-infected PBMCs results in increased PINCH, hp-Tau and CHIP.
A) Representative Western blot showing levels of PINCH1, hpTau, total Tau, Hsp90, Hsp70 and CHIP from neurons grown in media without PBMCs (con), neurons exposed to supernatant from control PBMCs without HIV infection (PBMC), supernatant from PBMCs infected with HIV (PBMC+HIV), or neurons exposed to cell-free virus (HIV). Grb-2 was used as the loading control. B) Fold change in hpTau/Total Tau.
Figure 3.
Reciprocal immunoprecipitations indicate PINCH interacts with hp-Tau and CHIP.
Neuronal lysate was immunoprecipitated with anti PINCH and reacted with antibodies against hp-Tau A) AT100 and B) AT8. C) Neuronal lysate was immunoprecipitated with anti-AT8 antibody and reacted with anti-PINCH, anti-Hsp90, anti-Hsp70 and CHIP. D) Neuronal lysate was immunoprecipitated with anti-PINCH antibody and reacted with anti-Hsp90, anti-Hsp70, anti-CHIP and anti-ILK. E) Reciprocal IP with anti-CHIP and reaction with anti-PINCH. F) Neuronal lysate was treated with (+) or without (−) phosphatase and immunoprecipitated with anti-HT7 antibody against total Tau and reacted with anti-PINCH antibody. G) The same neuronal lysate with (+) and without (−) phosphatase was immunoblotted with anti-HT7 and anti-PINCH antibodies without immunoprecipitation. Arrows indicate immunoreactive bands. Hc, heavy chain; lc, light chain; No IP (lysate), beads only (IgG); immunoprecipitation (IP).
Figure 4.
hp-Tau interacts with the first and second LIM domains of PINCH.
A) LIMS-specific deletion mutations predict that LIM1 and 2 domains of PINCH1 bind to hp-Tau. Expression plasmids for mutants (ΔLIMS 1–5) and full-length PINCH each with a FLAG-tag were generated via a PCR based strategy. Neuronal lysates were immunoprecipitated with anti-FLAG antibody and reacted with A) anti-hp-Tau (AT8), B) Nck-2 or E) MAP2 antibodies. Arrows indicate immunoreactive bands. FL, full length; ΔLIM, delta LIM indicates the LIM domain that was deleted; FLAG, epitope N-DYKDDDDK-C tag; Nck2, cytoplasmic adaptor protein that interacts with PINCH LIM4; MAP2, microtubule associated protein-2.
Figure 5.
Silencing PINCH during hp-Tau induction results in less hp-Tau accumulation.
A) Representative Western blots of neurons uninfected (con), infected with shRNA against PINCH1 and 2 (shRNA P1/2) or non-target control shRNA with and without okadaic acid (OA) treatment. B) Quantification of protein expression levels of hp-Tau (AT8), and C) PINCH relative to loading control (GAPDH). No changes were detected in Hsp90, Hsp70 or CHIP. Results are from 4 separate experiments and are expressed as fold change over uninfected neurons (con). * p<0.005, **p<0.001 by one-way ANOVA with Tukey-Kramer post-hoc analyses.
Figure 6.
Expression levels of hp-Tau and PINCH protein in frontal cortex brain tissue from patients.
A) Representative Western blot showing PINCH, soluble hp-Tau (s262) and total Tau (HT7) in age-matched controls (normal), AD Braak stages 1, 3, 5 (Br1, 3, 5), and HIV encephalitis (HIVE) patients. B) Graphic representation of percent total Tau that is hyperphosphorylated (hpTau/total Tau) in each case compared to control. C-G) Double immunofluorescence labeling of a frontal cortex tissue from a representative AD patient (Braak stage 3). C) from top left, panel shows DAPI for nuclei in blue; top right, PINCH in green; lower left, hp-Tau (s396) in red; lower right, co-localization of PINCH and hp-Tau. Arrowheads, arrows and asterisks indicate the same area in the tissue section. D) Double-immunolabeling of PINCH (green) and hp-Tau (red), co-localization, yellow; E) PINCH (green) and CHIP (red), co-localization (yellow); F) PINCH (green) and Hsp70 (red), co-localization (yellow); G) PINCH (green) and Hsp90 (red), no co-localization; H) Brain homogenate from a representative AD patient (Braak stage 3) immunoprecipitated with anti-PINCH antibody and reacted with anti-hp-Tau (AT8) antibody. Arrows, arrowheads show immunoreactive bands indicating PINCH-hp-Tau interaction.
Figure 7.
Expression levels of hp-Tau and PINCH in brain tissue from human Tau transgenic mouse.
A) Western analyses of anterior frontal cortex (Ant-Fc), ventro-lateral posterior cortex (V-L-post-FC), posterior frontal cortex (post-FC), cerebellum (CB). B) Double immunofluorescence labeling of PINCH (green) and hp-Tau (red), co-localization (yellow) in hippocampal tissue from a wild-type mouse and the Tau-Tg mouse and respectively.
Figure 8.
PINCH levels in relation to changes in Tau solubility.
The proteins from frontal cortex homogenates from normal, different Braak stages (1, 3, 5) of Alzheimer's disease (AD), HIV encephalitis (HIVE), and frontotemporal dementia (FTD) patients were fractionated into RAB (most soluble, RB), RIPA (less soluble, RB) and formic acid (FA) (least soluble). Representative Western blots from A) normal control, AD, HIVE, and FTD showing levels of PINCH, hpTau (s396) and total Tau (HT7). B) Coomassie stained gel of the same cases indicating total protein in each fraction. Compared to the control, increased levels of hp-Tau and PINCH are observed in disease cases. Loss of Tau and PINCH solubility are apparent in AD, HIVE and FTD, as well. Arrow in the FTD case indicates a blank lane in the gel.