Neurons that overexpress Par-4 exhibit enhanced mitochondrial dysfunction after mechanical injury compared to wild-type neurons. A. Rhd123 fluorescence (a measure of mitochondrial transmembrane potential) was measured before injury and at specific time intervals in both wild-type (top left) and Par-4 transgenic neurons (bottom left). Fluorescence intensity diminishes after injury, indicating the mitochondrial membrane potential has been compromised. B. Graph shows the mean average pixel intensity at each time point before and after injury. Par-4 transgenic neurons have significantly lower fluorescence intensity, beginning immediately after mechanical injury, when compared to wild-type neurons. Values are the means and standard derivations of determinations from eight separate cultures (20-30 neurons analyzed per culture). ***p<0.001 compared with corresponding values in cells from wild-type mice. ANOVA with Scheffe's post-hoc tests.

Par-4 overexpression in cortical neurons facilitates an earlier activation of caspase-3, contributing to a quicker progression through cell death. A. Levels of DEVD fluorescence (a measure of caspase-3 activation) were measured before injury and at specific time points after injury. Caspase-3 becomes activated after mechanical injury in both wild-type (top left) and Par4 transgenic neurons (bottom left). B. Quantification of DEVD fluorescence shows a higher degree of caspase-3 activation at each time point in Par-4 transgenic neurons compared to that in wild-type neurons, except at 24 hrs, when essentially all neurons overexpressing Par-4 have perished. Values are the means and standard derivations of determinations from eight separate cultures (20-30 neurons analyzed per culture). ***p<0.001. ANOVA with Scheffe's post-hoc tests.

Caspase-3 activation is significantly greater in transgenic mice that overexpress Par-4 compared to wild-type mice. A. Coronal sections were immunostained with the mouse caspase-3 primary antibody, which only recognizes and labels the cleaved, activated form of caspase-3. Caspase-3 activation was measured in sham-operated mice and at 8, 12, and 24 hr intervals after weight drop. Caspase-3 activation increases at each time point in the ipsilateral cortex (Ips), while activation in the contralateral cortex (Ctl) remains unchanged in both wild-type (top left) and Par-4 transgenic mice (bottom left). B. Graph shows a higher degree of caspase-3 activation in Par-4 transgenic mice following cortical impact in the ipsilateral cortex (Ips), but not in the contralateral cortex (Ctl). Values are the means and standard derivations of determinations made from coronal brain sections through the area of impact and adjacent to the area of impact. ***p<0.001 compared with corresponding values in cells from wild-type mice. ANOVA with Scheffe's post-hoc tests.

Neurons that overexpress Par-4 show an exacerbated rate of cell death following the mechanical insult compared to wild-type neurons. A. Representative Western blotting analysis showing high level expression of Par-4 protein in cortical neurons from transgenic mice. Primary cultures of cortical neurons were established and western blot analyses were performed in 7-day old cultures using a monoclonal antibody raised against a recombinant protein corresponding to amino acids 1-334 representing full length PAR4 of rat origin (Santa Cruz Biotechnology, Inc.). Relatively high levels of Par-4 protein were detected in neurons from Par-4 transgenic mice (lane 2), while only minimal levels of endogenous Par-4 were observed in normal neurons from wild-type mice (lane 1). B. Genotyping of Par-4 transgenic mice using a PCR-based protocol. We have developed a quick PCR-based assay of the DNA from tail biopsies to amplify a 236-bp simian virus 40 fragment from pNSE-Par4 vector, which is detectable only in mice transgenic for Par-4 (lanes 1-3), but not in wild-type mice (lanes 4-6). C. Phase contrast images of cortical neurons from wild-type and Par-4 transgenic mice before and after injury. Overall neuron survival decreases at each time point. D. Graph shows percentage of surviving cells at each time point as indicated: uninjured (Un), immediately after injury (0), 4, 8, 12 and 24 hrs after injury. A higher percentage of cell death was observed in Par-4 transgenic cortical neurons compared to wild-type neurons. Values are the means and standard derivations of determinations from eight separate cultures (20-30 neurons analyzed per culture). ***p<0.001 compared with corresponding values in cells from wild-type mice. ANOVA with Scheffe's post-hoc tests.

Cortical neurons overexpressing Par-4 show an increased accumulation of superoxide and hydrogen peroxide following mechanical injury compared to wild-type neurons. A. HE fluorescence (a measure of superoxide levels) and C. DCF fluorescence (a measure of hydrogen peroxide levels) were measured before injury and 12 hrs post injury in both wild-type (top) and Par-4 transgenic neurons (bottom). Fluorescence intensity of HE and DCF increases 12 hrs post injury in wild-type and Par-4 transgenic cortical neurons. B. Bar graph shows a higher accumulation of superoxide in neurons that overexpress Par-4 compared to wild-type neurons. D. Similar results are observed for the production of hydrogen peroxide in neurons overexpressing Par-4 when compared to wild-type neurons. Values are the means and standard derivations of determinations from eight separate cultures (20-30 neurons analyzed per culture). ***p<0.001 compared with corresponding values in cells from wild-type mice. ANOVA with Scheffe's post-hoc tests.

Traumatic brain injury is exacerbated in Par-4 transgenic mice compared to wild-type mice. A. Coronal sections through the damaged cerebral cortex of adult mice were stained with cresyl violet. Nissl staining was measured in sham-operated mice and at 8, 24, and 48 hr intervals after cortical impact. Cortical damage is observed in the ipsilateral cortex (Ips), while the contralateral cortex (Ctl) is unaffected in wild-type (top left) and Par-4 transgenic mice (bottom left). B. Graph shows the percentage of cortical lesion volume in ipsilateral (Ips) and contralateral (Ctl) sides of the cortical impact in both wild-type and Par-4 transgenic mice. A significantly larger cavity was produced in the ipsilateral cortex (Ips) by 8 hrs and more prominently by 24 and 48 hrs after cortical impact in mice that overexpress Par-4 when compared to wild-type mouse brains. The damaged portion of the cortex was characterized by faint or lack of staining and was clearly delineated from healthy brain tissue. The amount of cortical damage is expressed as a percentage of total cortical volume, calculated using the Cavalieri principle (see Materials and Methods). Values are the means and standard derivation of determinations made from coronal brain sections through the area of impact and the contralateral side of impact. *p<.0.05, ***p<0.001 compared with corresponding values in sections from wild-type mice. ANOVA with Scheffe's post-hoc tests.