Long-term accumulation of amyloid-beta, beta-secretase, presenilin-1, and caspase-3 in damaged axons following brain trauma - PubMed (original) (raw)

Comparative Study

Long-term accumulation of amyloid-beta, beta-secretase, presenilin-1, and caspase-3 in damaged axons following brain trauma

Xiao-Han Chen et al. Am J Pathol. 2004 Aug.

Abstract

Plaques composed of amyloid beta (Abeta) have been found within days following brain trauma in humans, similar to the hallmark plaque pathology of Alzheimer's disease (AD). Here, we evaluated the potential source of this Abeta and long-term mechanisms that could lead to its production. Inertial brain injury was induced in pigs via head rotational acceleration of 110 degrees over 20 ms in the coronal plane. Animals were euthanized at 3 hours, 3 days, 7 days, and 6 months post-injury. Immunohistochemistry and Western blot analyses of the brains were performed using antibodies specific for amyloid precursor protein (APP), Abeta peptides, beta-site APP-cleaving enzyme (BACE), presenilin-1 (PS-1), caspase-3, and caspase-mediated cleavage of APP (CCA). Substantial co-accumulation for all of these factors was found in swollen axons at all time points up to 6 months following injury. Western blot analysis of injured brains confirmed a substantial increase in the protein levels of these factors, particularly in the white matter. These data suggest that impaired axonal transport due to trauma induces long-term pathological co-accumulation of APP with BACE, PS-1, and activated caspase. The abnormal concentration of these factors may lead to APP proteolysis and Abeta formation within the axonal membrane compartment.

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Figures

Figure 1

Figure 1

Representative photomicrographs demonstrating APP and NF protein accumulation in damaged axons in the subcortical white matter and basal ganglia detected by antibodies to 22C11/APP N-terminal and N52/NF200 at 3, 7 days, and 6 months following injury in the pig. Dark staining represents immunoreactivity of APP and NF proteins in swollen axons. Bar, 25 μm.

Figure 2

Figure 2

Representative photomicrographs revealing a modest number of pyknotic neurons in CA1 and CA3 subfields of the hippocampus (top panels) and foamy macrophage infiltrations within subcortical white matter, basal ganglia and lining blood vessels. Axonal bulbs were close to macrophage infiltrations (black arrows), detected by antibody OX42 at 7 days and 6 months post-injury (bottom panels). Bar, 25 μm.

Figure 3

Figure 3

Representative photomicrographs revealing Aβ accumulation in axonal bulbs in the basal ganglia at 3, 7 days, and 6 months after brain injury in the pig. Aβ was identified by antibodies to 4G8, BCO5, 6F/3D, Aβ1–40, 10Δ5. Bar, 25 μm.

Figure 4

Figure 4

Representative photomicrographs demonstrating BACE, PS1 and CCA in damaged axons in the subcortical white matter and basal ganglia at 3 days to 6 months following brain injury in the pig. Accumulations in axons were detected by antibodies BACE-2, PS-1, and 249. Bar, 25 μm.

Figure 5

Figure 5

Representative photomicrographs showing Aβ-containing plaque-like profiles in the gray and white matter. Occasionally, perivascualr Aβ depostis were found (top, middle). Aβ accumulation was also found in cortical and cerebellar neurons at 3 days to 6 months following brain trauma. Aβ was identified by antibodies, 6F/3D, Aβ1–40, 13335. Bar, 25 μm.

Figure 6

Figure 6

Representative double-immunofluorescence photomicrographs demonstrating co-accumulations of proteins in damaged axons (A–L), neurons (M–N) and macrophages (O–P) at 3 days and 6 months post-injury. Merged green and red fluorescence shown in yellow. In axon bulbs in the white matter, co-accumulation Aβ (antibodies 6F3D and 13335/Green) was found with CCA (249/Red) in (A) and (F), caspase-3 (P20/Red) in (B), BACE (BACE-2/Red) in (G), APP (22C11/Red) in (E), and PS-1 (PS-1/Red) in (H). Co-accumulation of BACE (Green) was found with APP (Red) in (C) and (I), kinesin (L1/Red) in (D) and (K), and CCA (Red) in (J). Co-accumulation of APP (Red) was found with PS-1 (Green) in (L). In neurons, Aβ (Green) co-accumulated with APP (Red) in (M) and CCA (Red) in (N). Macrophages demonstrated co-immunoreactivity of Aβ (13335/Green) with OX42 (CD11b/Red) in (O) and (P). Bar, 25 μm.

Figure 7

Figure 7

Representative photomicrographs showing Congo red staining in damaged axons in subcortical white matter at 3 days, 7 days and 6 months post-injury (A, C, D, E, and F), and in plaque-like profiles in the white matter at 3 and 7 days post-injury (B and D). Congo red stained plaque-like profiles (D, downward arrow) could be found in proximity with axonal bulbs (D, diagonal arrow). Double-staining with Thioflavin S and an anti-Aβ antibody shows co-localization in swollen axons in the subcortical white matter (G–J) at 3 days to 6 months after brain trauma, as well as in a few plaque-like profiles in the gray matter (K) at 6 months following brain injury. Bar, 25 μm.

Figure 8

Figure 8

Western blot analysis of Aβ in injured and sham brain white matter. Immunoreactive bands at the molecular weight of Aβ peptides (approximately 4 kd) were found at all post-injury time points by all anti-Aβ antibodies used (primary antibodies listed below graphs). Graphic representation of quantitative analysis of the optical density of the bands used mean values and SE from two separate experiments. P values refer to comparison between sham and groups of injured animals at different time points. ***, P < 0.001.

Figure 9

Figure 9

Western blot analysis of APP C99 fragment (14 kd), BACE, and PS-1 in injured and sham brain white matter. Primary antibodies used are listed below graphs. Quantitative analysis of the protein expression is shown graphically, with mean values and SE of optical density of immunostaining from two separate experiments. P values refer to comparison between groups of injured animals and sham. *, P < 0.05; ***, P < 0.001; †, P < 0.05.

Figure 10

Figure 10

Western blot analysis of caspase-mediated APP proteolysis, caspase-3, and kinesins in injured and sham brain white matter. (A) Banding representing CCA, (B) the 20 kd large subunit of caspase-3 and procapase, (C) Kinesin-L, and (D) Kinesin-H. Primary antibodies listed below graphs. Quantitative analysis of the protein expression of CCA, caspase-3, and kinesins is shown graphically with mean values and SE of optical density of immunostaining from two separate experiments. P values refer to comparison between groups of injured animals at different time points and sham. *, P < 0.05; **, P < 0.01; ***, P < 0.001; †††, P < 0.001.

Figure 11

Figure 11

Proposed pathway of Aβ production and dispersal in diffuse axonal injury.

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