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Aging and disease    2016, Vol. 7 Issue (3) : 267-277     DOI: 10.14336/AD.2015.1020
Original Article |
Proteolytic Cleavage of Apolipoprotein E in the Down Syndrome Brain
Day Ryan J.1, McCarty Katie L.2, Ockerse Kayla E.1, Head Elizabeth2, Rohn Troy T.1
1Department of Biological Sciences, Boise State University, Boise, Idaho, 83725, USA
2Department of Pharmacology& Nutritional Sciences, Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, 40536, USA
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Down syndrome (DS) is one of the most common genetic causes of intellectual disability and is characterized by a number of behavioral as well as cognitive symptoms. Many of the neuropathological features of early-onset Alzheimer’s disease (AD) including senile plaques and neurofibrillary tangles (NFTs) are also present in people with DS as a result of triplication of the amyloid precursor gene on chromosome 21. Evidence suggests that harboring one or both apolipoprotein E4 (APOE4) alleles may increase the risk for AD due to the proteolytic cleavage of apoE4 and a subsequent loss of function. To investigate a role for the apoE proteolysis in vivo, we compared three autopsy groups; 7 DS with AD neuropathology cases over 40 years, 5 young DS cases without AD pathology under 40 years (YDS) and 5 age-matched control cases over 40 years by immunohistochemistry utilizing an antibody that detects the amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE fragment antibody (nApoECF) revealed labeling of pyramidal neurons in the frontal cortex of YDS cases, whereas in the DS-AD group, labeling with nApoECF was prominent within NFTs. NFT labeling with nApoECF was significantly greater in the hippocampus versus the frontal cortex in the same DS-AD cases, suggesting a regional distribution of truncated apoE. Colocalization immunofluorescence experiments indicated that 52.5% and 53.2% of AT8- and PHF-1-positive NFTs, respectively, also contained nApoECF. Collectively, these data support a role for the proteolytic cleavage of apoE in DS and suggest that apoE fragmentation is closely associated with NFTs.

Keywords Alzheimer’s disease      beta-amyloid      paired helical filaments      proteolysis      neurofibrillary tangles      immunohistochemistry     
Corresponding Authors: Rohn Troy T.   
About author:

These authors equally contribute this work

Issue Date: 09 January 2016
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Day Ryan J.
McCarty Katie L.
Ockerse Kayla E.
Head Elizabeth
Rohn Troy T.
Cite this article:   
Day Ryan J.,McCarty Katie L.,Ockerse Kayla E., et al. Proteolytic Cleavage of Apolipoprotein E in the Down Syndrome Brain[J]. Aging and disease, 2016, 7(3): 267-277.
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CaseNPDSexPMIAgeRegionCause of DeathAPOE Genotype
1NormalF2446Frontal CortexMultiple injuriesN/A
2NormalF2151Frontal CortexCardiovascular diseaseN/A
3NormalM1757Frontal CortexArteriosclerotic cardiovascular diseaseN/A
4NormalM565Frontal CortexCardiac arrestN/A
5NormalM367Frontal CortexCardiomyopathyN/A
6YDSM2424Frontal CortexCardiac arrhythmiaN/A
7YDSM431Frontal CortexPneumoniaN/A
8YDSF3634Frontal Cortex and HippocampusSeptic shockN/A
9YDSF1239Frontal Cortex and HippocampusCancerN/A
10YDSM533Frontal Cortex and HippocampusAcute bronchopneumoniaN/A
11DS-ADM646Frontal cortex and HippocampusCardiac respiratory failure2/3
12DS-ADM1856Frontal cortex and HippocampusAlzheimer’s disease3/3
13DS-ADF557Frontal cortex and HippocampusOther3/3
14DS-ADM1066Frontal CortexCongestive heart failureN/A
15DS-ADM267Frontal CortexPneumoniaN/A
16DS-ADF357HippocampusAlzheimer’s disease3/3
Table 1  Case Demographics
Figure 1.  Localization of an amino-terminal fragment of apoE in the frontal cortex of Down’s syndrome

(A-B): Application of the nApoECF antibody in frontal cortex tissue sections from YDS cases revealed labeling predominantly within pyramidal neurons (arrows, low magnification Panel A) and (arrows, high magnification Panel B). (C): In contrast, in DS-AD cases, in addition to neuronal staining, more mature, fibrillary NFTS were labeled with the nApoECF antibody (arrows), whereas little immunoreactivity was observed in age-matched control cases (D). (E): A least significant difference post hoc test analysis of the number of nApoECF-positive neurons and NFTs in frontal cortex indicated a significant difference between DS-AD cases and age-matched controls (p= 0.02) and YDS groups (p = 0.05). For all three groups, n = 5, ±S.E.M. All scale bars represent 10 µm except for Panel D, which represents 50 µm.

Figure 2.  Localization of an amino-terminal fragment of apoE in the hippocampus of Down’s syndrome

Application of the nApoECF antibody in hippocampal tissue sections revealed very little labeling in YDS cases (A) or in age-matched controls (C), however, strong immunolabeling of NFTs was observed in DS-AD cases (B). (D): Three DS-AD cases were quantified (±S.D.) for the number of nApoECF-positive NFTs comparing hippocampal versus frontal cortex regions. The data revealed a clear difference between the numbers of labeled NFTs between the two regions with the hippocampus giving consistently higher numbers. Case #1, #2, and #3 correspond to subjects 13, 12, and 11, respectively, as listed in Table 1. All scale bars for Panels A and C represent 50 µm and 10 µm for Panel D.

Figure 3.  Co-localization of an amino-terminal fragment of apoE within NFTs in hippocampal tissue sections of the Down’s syndrome brain

(A-C): Representative confocal immunofluorescence double-labeling utilizing the nApoECF antibody (green, Panel A) and PHF-1 (red, Panel B) revealed strong co-localization of the two antibodies within a NFT located in the hippocampus (Panel C). (D-F): Identical to Panels A-C with the exception of AT8 (red) being employed. For Panels A-F, images were captured from the CA1 region of the hippocampus in representative DS-AD cases. (G and H): Quantification of NFTs double-labeled by PHF-1, AT8, and nApoECF. For both panels, data show the number of NFTs labeled with nApoECF alone (blue bar), AT8 (G) or PHF-1 (H) alone (green bars) or those NFTs that were labeled with both antibodies (red bars). NFTs were identified in a 20X field within hippocampal tissue sections by immunofluorescence overlap microscopy (n=3 fields using three different DS cases) ±S.E.M. For Panel G there were no statistical differences between the various test groups. Data indicated that roughly 52.5% of AT8-positive NFTs also contained nApoECF, whereas 53.2% of all identified PHF-1-positive NFTs were labeled with nApoECF.

Figure 4.  Co-localization of nApoECF with a full-length amino-terminal antibody to apoE4 in NFTs of the Down Syndrome brain

(A-C): Representative confocal immunofluorescence double-labeling in CA1 region of the hippocampus in DS-AD cases utilizing an antibody that recognizes the amino-terminal region of full-length apoE4 (green, A) and nApoECF (red, B), with the overlap image shown in Panel C. Strong co-localization was indicated in both apparent NFTs and blood vessels (yellow, C). (D): In contrast, no co-localization with nApoECF was observed in a similar experiment using a full-length antibody to the C-terminal end of apoE4, which strongly labeled blood vessels (green, D), but not NFTs (arrows, D).

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