Increased Amyloid Precursor Protein and Tau Expression Manifests as Key Secondary Cell Death in Chronic Traumatic Brain Injury

Abstract

In testing the hypothesis of Alzheimer's disease (AD)-like pathology in late stage traumatic brain injury (TBI), we evaluated AD pathological markers in late stage TBI model. Sprague-Dawley male rats were subjected to moderate controlled cortical impact (CCI) injury, and 6 months later euthanized and brain tissues harvested. Results from H&E staining revealed significant 33% and 10% reduction in the ipsilateral and contralateral hippocampal CA3 interneurons, increased MHCII-activated inflammatory cells in many gray matter (8-20-fold increase) and white matter (6-30-fold increased) regions of both the ipsilateral and contralateral hemispheres, decreased cell cycle regulating protein marker by 1.6- and 1-fold in the SVZ and a 2.3- and 1.5-fold reductions in the ipsilateral and contralateral dentate gyrus, diminution of immature neuronal marker by two- and onefold in both the ipsilateral and contralateral SVZ and dentate gyrus, and amplified amyloid precursor protein (APP) distribution volumes in white matter including corpus callosum, fornix, and internal capsule (4-38-fold increase), as well as in the cortical gray matter, such as the striatum hilus, SVZ, and dentate gyrus (6-40-fold increase) in TBI animals compared to controls (P's < 0.001). Surrogate AD-like phenotypic markers revealed a significant accumulation of phosphorylated tau (AT8) and oligomeric tau (T22) within the neuronal cell bodies in ipsilateral and contralateral cortex, and dentate gyrus relative to sham control, further supporting the rampant neurodegenerative pathology in TBI secondary cell death. These findings indicate that AD-like pathological features may prove to be valuable markers and therapeutic targets for late stage TBI. J. Cell. Physiol. 232: 665-677, 2017. © 2016 Wiley Periodicals, Inc.

Figure 1

Reduced cell proliferation, impaired neurogenesis, and increased hippocampal cell loss in late stage TBI. Part A, quantitative stereological analysis revealed significant decrements in cell proliferation and neurogenesis in the SVZ and DG after late stage TBI compared to sham animals (*P's < 0.05). Part A, bottom right, quantitative analyses of total number of CA3 neurons revealed a significant increase in neuronal cell loss after TBI compared to sham animals (*P's > 0.05). Part B, top, photomicrographs are representative coronal brain sections of the corresponding ipsilateral and contralateral side of the SVZ, and DG regions stained with a cell proliferation marker (Ki67), an immature neuronal marker (DCX) at 6 months post TBI of sham, and TBI animals. Arrows denote positive staining. Part B, bottom, photomicrographs are representative coronal brain sections staining with H&E from ipsilateral and contralateral CA3 area of the hippocampus of sham, and TBI animals. Arrows denote dark pink/purple cells characteristic of shrunken and condensed nuclei and hypereosinophilic cytoplasm, and neuronal cell loss within the CA3 region to the contralateral and ipsilateral side respectively in TBI animals. Scale bar: 50 μm. *P < 0.05; P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant. Ki67 and DCX data are expressed as estimated # of positive cells. H&E data are expressed as total # of cells. Data are expressed as mean ± SEM.

Figure 2

Increased MHCII+ activated cells in proximal and remote gray matter areas in late stage TBI. Part A, stereological analysis of MHCII+ cells estimated volume in cortex, striatum, DG, and SVZ revealed significant upregulation of activated MHCII+ cells in the ipsilateral side of TBI animals compared to their contralateral side across all gray matter areas analyzed (P's < 0.0001), except hilus and thalamus (P > 0.05). There were significant upregulations of activated MHCII+ cells in both ipsilateral and contralateral gray matter areas of TBI animals (P's < 0.0001) compared to sham animals (P's < 0.05). Part B, photomicrographs are representative coronal brain sections showing gray matter areas ipsilateral to injury stained with M1 activated immune cells marker (MHCII) 6 months post TBI injury. Arrows indicate positive staining for activated MHCII+ cells in cortex, striatum, DG, hilus, SVZ, and thalamus. Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. MHCII data are expressed as estimated volume of positive cells. Data are expressed as mean ± SEM.

Figure 3

Increased MHCII+ activated cells in proximal and remote white matter areas in late stage TBI. Part A, stereological analysis of MHCII+ cells estimated volume in corpus callosum, fornix, and internal capsule revealed no significant differences of activated MHCII+ cells in the ipsilateral side of TBI animals compared to their contralateral side across all white matter areas analyzed (P's > 0.05). There were significant upregulation of activated MHCII+ cells in both ipsilateral and contralateral side of TBI animals (P's < 0.0001) across all white matter areas analyzed compared to sham animals (P's < 0.05). Part B, photomicrographs are representative coronal brain sections showing white matter areas ipsilateral to injury stained with M1 activated immune cells marker (MHCII) 6 months post TBI injury. Arrows indicate positive staining for activated MHCII+ cells in corpus callosum, fornix, and internal capsule. Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. MHCII data are expressed as estimated volume of positive cells. Data are expressed as mean ± SEM. Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Data are expressed as mean ± SEM.

Figure 4

Overexpression of intra‐neuronal APP in proximal and remote gray matter areas in late stage TBI. Part A, stereological analysis of APP+ neurons estimated volume in cortex, striatum, DG, and hilus revealed significant increase of APP+ neurons in the ipsilateral side of TBI animals compared to their contralateral side across all gray matter areas analyzed (P's < 0.0001), except SVZ and thalamus (P > 0.05). There was significant overexpression of APP+ expressing neurons in both ipsilateral and contralateral gray matter areas of TBI animals (P's < 0.0001) compared to sham animals (P's < 0.05), except DG, hilus, SVZ, and thalamus (P's > 0.05). Part B, photomicrographs are representative coronal brain sections showing gray matter areas ipsilateral to injury stained with APP marker post TBI injury. Arrows indicate overexpression of intra‐neuronal APP staining in cortex, striatum, DG, hilus, and SVZ. Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. APP data are expressed as estimated volume of positive cells. Data are expressed as mean ± SEM.

Figure 5

Overexpression of APP in proximal and remote white matter areas in late stage TBI. Part A, stereological analysis of APP estimated volume in corpus callosum, fornix, and internal capsule revealed significant overexpression of APP+ staining in the white matter ipsilateral side of TBI animals compared to their contralateral side across all areas analyzed (P's < 0.0001). There was significant overexpression of APP+ staining in both ipsilateral and contralateral white matter areas of all TBI animals (P's < 0.0001) compared to sham animals (P's < 0.0001). Part B, photomicrographs are representative coronal brain sections showing white matter areas proximal and remote from TBI injury and ipsilateral to injury stained with APP marker post TBI injury. Arrows indicate overexpression of APP staining in corpus callosum, fornix, and internal capsule. Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. APP data are expressed as estimated volume of positive cells. Data are expressed as mean ± SEM.

Figure 6

TBI‐induced tau phosphorylation at Ser202/Thr205 (AT8) in the cortex and dentate gyrus (DG) of the hippocampus 6 months post TBI. Part A and C, quantitative analysis of phosphorylated tau (AT8) expression in the cortex and DG revealed significant increase of phosphorylated tau in the ipsilateral and contralateral side of TBI animals compared to sham animals (P's < 0.0001). Part B and D, confocal immunofluorescent images of phosphorylated tau (AT8) (green) and DAPI (blue) show positive expression in cortex and DG of late TBI injury. Arrows indicate positive expression of phosphorylated tau in the cell body of cortical and granular neurons. Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Phosphorylated tau (AT8) data are expressed as estimated staining intensity from positive cells. Data are expressed as mean ± SEM.

Figure 7

TBI‐induce accumulation of oligomeric tau (T22) in the cortex and dentate gyrus (DG) of the hippocampus 6 months post TBI. Part A and C, quantitative analysis of oligomeric tau (T22) accumulation in the cortex and DG revealed significant increase of in the ipsilateral and contralateral side of TBI animals compared to sham animals (P's < 0.0001). Part B and D, confocal immunofluorescent images of oligomeric tau (T22) (red) and DAPI (blue) show positive expression in throughout cortex and in the soma of neurons from the DG. Arrows indicate positive expression of aggregates of oligomeric tau in the cortex and granular neurons in the DG. Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Oligomeric tau (T22) data are expressed as estimated staining intensity from positive cells. Data are expressed as mean ± SEM.