Subacute Changes in Cleavage Processing of Amyloid Precursor Protein and Tau following Penetrating Traumatic Brain Injury

<div><p>Traumatic brain injury (TBI) is an established risk factor for the development of Alzheimer’s disease (AD). Here the effects of severe penetrating TBI on APP and tau cleavage processing were investigated in a rodent model of penetrating ballistic-like brain injury (PBBI). PBBI was induced by stereotactically inserting a perforated steel probe through the right frontal cortex of the anesthetized rat and rapidly inflating/deflating the probe’s elastic tubing into an elliptical shaped balloon to 10% of total rat brain volume causing temporary cavitation injury. Separate animals underwent probe injury (PrI) alone without balloon inflation. Shams underwent craniectomy. Brain tissue was collected acutely (4h, 24h, 3d) and subacutely (7d) post-injury and analyzed by immunoblot for full length APP (APP-FL) and APP beta c-terminal fragments (βCTFs), full length tau (tau-FL) and tau truncation fragments and at 7d for cytotoxic Beta amyloid (Aβ) peptides Aβ40 and Aβ42 analysis. APP-FL was significantly decreased at 3d and 7d following PBBI whereas APP βCTFs were significantly elevated by 4h post-injury and remained elevated through 7d post-injury. Effects on βCTFs were mirrored with PrI, albeit to a lesser extent. Aβ40 and Aβ42 were significantly elevated at 7d following PBBI and PrI. Tau-FL decreased substantially 3d and 7d post-PBBI and PrI. Importantly, a 22 kDa tau fragment (tau22), similar to that found in AD, was significantly elevated by 4h and remained elevated through 7d post-injury. Thus both APP and tau cleavage was dramatically altered in the acute and subacute periods post-injury. As cleavage of these proteins has also been implicated in AD, TBI pathology shown here may set the stage for the later development of AD or other tauopathies.</p></div

Pilot study: tau22 fragmentation in regions of interest.

<p>Tissue from specific regions of interest (Frontal Cortex, Striatum, Hippocampus, and Residual Midbrain) were qualitatively evaluated by Western blot for the presence or absence of tau22 fragmentation (A) 24h following PBBI (n = 3) and (B) 1m following PrI and PBBI (n = 3).</p

Increased Aβ40 and Aβ42 7 days post-TBI.

<p>Amyloid levels from Sham, PrI and PBBI were evaluated by chemiluminescent ELISAs 7d post-injury. (A) Quantification of Aβ40 (B) Representative visualizing of Aβ40 chemiluminescent ELISA wells. (C) Quantification of Aβ42 (D) Representative visualizing of Aβ42 chemiluminescent ELISA wells. Both Aβ40 and Aβ42 were significantly upregulated by PrI and PBBI 7d post-injury with PBBI inducing significantly more Aβ40 and Aβ42 than PrI. Statistical analysis of changes was conducted (K-W, * p<0.05; error bars: standard error of the mean, n = 10 per group).</p

Detection of FL <i>tau</i> and <i>tau</i> fragments following penetrating TBI.

<p>Protein levels from Sham, PrI and PBBI were evaluated by Western blot at time points 4h, 24h, 3d and 7d post injury. Bands were measured and normalized to corresponding βactin levels. A representative blot at 7d post injury is shown in (A). Quantification corresponding to (B) tau-FL, (C) 40 kDa tau and (D) 22 kDa tau show significant loss of tau-FL and 40 kDa tau and significant increases in 22 kDa tau fragment through 7d. Statistical analysis of changes at each time point was conducted (K-W, * p<0.05, 2 way ANOVA # p<0.05; error bars: standard error of the mean, n = 8–10 per group).</p

Epitope binding confirmed for Tau-FL and Tau22 using Tau1 and Tau5 antibodies.

<p>Specific binding to proline rich domain of tau confirmed for Tau-FL and tau22 fragment in samples 24h post-injury using (A) Tau1 and (B) Tau5 antibodies against total tau and compared to positive control transgenic AD model mouse brain lysate (3XTg-AD). The 22 kDa tau fragment (*) was detected by both total tau antibodies. Experiment replicated 3 times for (sham, PBBI).</p

22 kDa Tau fragment binding confirmation.

<p>Protein levels from 7d post-injury PBBI brain lysate and 7d post-PBBI brain lysate that underwent immunoprecipitation with Tau 1 antibody (tau IP) were evaluated for evidence of (A) tau caspase cleavage at Asp421and (B) tau phosphorylation using AT8 antibody. Evidence of caspase cleavage of the 22 kDa tau fragment was seen using the tau Asp421 specific caspase antibody in both the original brain lysate as well as the Tau 1 immunoprecipitation product. Antibody AT8, specific for phosphorylation at sites S202, did not detect the 22 kDa tau fragment. Experiments replicated at least 3 times (sham, PBBI). AT8 phosphorylation experiments replicated twice (sham, PBBI).</p

Loss of APP and accumulation of βCTFs after TBI.

<p>Protein levels from sham, PrI and PBBI were evaluated by Western blot at time points 4h, 24h, 3d and 7d days post injury. Bands were measured and normalized to corresponding βactin levels. A representative blot at 3d post injury is shown in (A). Quantification corresponding to (B) APP-FL, (C), APP βCTFs, and (D) APP αCTFs show significant loss of APP-FL with PBBI and significant increases in βCTFs at all time-points for PBBI and at 7d for PrI. APP αCTFs were significantly increased post-PrI and -PBBI at 24h and significantly decreased at 3d post-PBBI Statistical analysis of changes at each time point was conducted (K-W, * p<0.05, 2 way ANOVA # p<0.05; error bars: standard error of the mean, n = 8–10 per group).</p

Domain location of antibodies Tau1, 5E2, Tau5 and caspase cleavage at Asp421.

<p>Domain location of antibodies Tau1, 5E2, Tau5 and caspase cleavage at Asp421.</p

PBBI model (A) Visualization of PBBI probe (B) PBBI probe with balloon inflation (C) anatomical location of injury tract with probe alone and probe with balloon inflation.

<p>PBBI model (A) Visualization of PBBI probe (B) PBBI probe with balloon inflation (C) anatomical location of injury tract with probe alone and probe with balloon inflation.</p