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Apolipoprotein-E forms dimers in human frontal cortex and hippocampus

Elliott, David A, Halliday, Glenda M, Garner, Brett·2010·BMC Neuroscience
DOI 10.1186/1471-2202-11-23PMC PMC2837047
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Abstract

Background: Apolipoprotein-E (apoE) plays important roles in neurobiology and the apoE4 isoform increases risk for Alzheimer's disease (AD). ApoE3 and apoE2 are known to form disulphide-linked dimers in plasma and cerebrospinal fluid whereas apoE4 cannot form these dimers as it lacks a cysteine residue. Previous in vitro research indicates dimerisation of apoE3 has a significant impact on its functions related to cholesterol homeostasis and amyloid-beta peptide degradation. The possible occurrence of apoE dimers in cortical tissues has not been examined and was therefore assessed. Human frontal cortex and hippocampus from control and AD post-mortem samples were homogenised and analysed for apoE by western blotting under both reducing and non-reducing conditions. Results: In apoE3 homozygous samples, ~12% of apoE was present as a homodimer and ~2% was detected as a 43 kDa heterodimer. The level of dimerisation was not significantly different when control and AD samples were compared. As expected, these dimerised forms of apoE were not detected in apoE4 homozygous samples but were detected in apoE3/4 heterozygotes at a level approximately 60% lower than seen in the apoE3 homozygous samples. Similar apoE3 dimers were also detected in lysates of SK-N-SH neuroblastoma cells and in freshly prepared rabbit brain homogenates. The addition of the thiol trapping agent, iodoacetamide, to block reactive thiols during both human and rabbit brain sample homogenisation and processing did not reduce the amount of apoE homodimer recovered. These data indicate that the apoE dimers we detected in the human brain are not likely to be post-mortem artefacts. Conclusion: The identification of disulphide-linked apoE dimers in human cortical and hippocampal tissues represents a distinct structural difference between the apoE3 and apoE4 isoforms that may have functional consequences.

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Figure 1 ApoE3 dimers are present in human hippocampus. The presence of disulphide-linked dimers of apoE were detected by analysing samples under both non-reducing "NR" and reducing "R" SDS-PAGE conditions. The susceptibility of dimers to thrombin cleavage was tested using three different conditions; storage at -80°C with no enzyme added "No enz", incubation at 37°C in the presence of thrombin "Thr" or heat-inactivated thrombin "hi-Thr". TBS-soluble fractions from control apoE3 (A), AD apoE3 (B) and AD apoE4 (C) homozygous hippocampal tissue samples were analysed. Western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (Con n = 1, AD n = 2) are identified according to the Case # code given in Table 1.
Figure 1 ApoE3 dimers are present in human hippocampus. The presence of disulphide-linked dimers of apoE were detected by analysing samples under both non-reducing "NR" and reducing "R" SDS-PAGE conditions. The susceptibility of dimers to thrombin cleavage was tested using three different conditions; storage at -80°C with no enzyme added "No enz", incubation at 37°C in the presence of thrombin "Thr" or heat-inactivated thrombin "hi-Thr". TBS-soluble fractions from control apoE3 (A), AD apoE3 (B) and AD apoE4 (C) homozygous hippocampal tissue samples were analysed. Western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (Con n = 1, AD n = 2) are identified according to the Case # code given in Table 1.CC BY · PubMed Central
Figure 2 ApoE3 dimers are present in both hippocampus and frontal cortex of control apoE 3/3 brain homogenates. Comparisons were made between the hippocampus and frontal cortex in each individual control apoE3/3 brain, under non-reduced (NR) and reduced (R) conditions. Western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (Con n = 5) are identified according to the Case # code given in Table 1.
Figure 2 ApoE3 dimers are present in both hippocampus and frontal cortex of control apoE 3/3 brain homogenates. Comparisons were made between the hippocampus and frontal cortex in each individual control apoE3/3 brain, under non-reduced (NR) and reduced (R) conditions. Western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (Con n = 5) are identified according to the Case # code given in Table 1.CC BY · PubMed Central
Figure 3 ApoE3 dimers are present in the detergent (Triton-X-100) soluble fraction of both control and AD brain homogenates. The presence of apoE3 dimers was assessed in the Triton-X-100 soluble fraction of frontal cortex samples from control apoE3/3 and AD apoE3/3 brains, under non-reduced (NR) and reduced (R) conditions. Western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (Con n = 2, AD n = 2) are identified according to the Case # code given in Table 1.
Figure 3 ApoE3 dimers are present in the detergent (Triton-X-100) soluble fraction of both control and AD brain homogenates. The presence of apoE3 dimers was assessed in the Triton-X-100 soluble fraction of frontal cortex samples from control apoE3/3 and AD apoE3/3 brains, under non-reduced (NR) and reduced (R) conditions. Western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (Con n = 2, AD n = 2) are identified according to the Case # code given in Table 1.CC BY · PubMed Central
Figure 4 ApoE dimers are absent in the AD apoE4/4 brain homogenates. The absence of apoE dimers in AD apoe4/4 brains was confirmed in all available samples (n = 7) and compared to a reference AD apoE3/3 brain. Samples were analysed under non-reduced (NR) and reduced (R) conditions and western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (AD n = 8) are identified according to the Case # code given in Table 1.
Figure 4 ApoE dimers are absent in the AD apoE4/4 brain homogenates. The absence of apoE dimers in AD apoe4/4 brains was confirmed in all available samples (n = 7) and compared to a reference AD apoE3/3 brain. Samples were analysed under non-reduced (NR) and reduced (R) conditions and western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (AD n = 8) are identified according to the Case # code given in Table 1.CC BY · PubMed Central
Figure 5 Proportion of apoE present as dimers is significantly lower in heterozygous apoE3/4 AD brain homogenates. The proportion of total apoE present as dimers was compared between homozygous apoE3/3 and heterozygous apoE3/4 frontal cortex homogenates, under non-reduced (NR) and reduced (R) conditions. Western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (AD n = 8) are identified according to the Case # code given in Table 1.
Figure 5 Proportion of apoE present as dimers is significantly lower in heterozygous apoE3/4 AD brain homogenates. The proportion of total apoE present as dimers was compared between homozygous apoE3/3 and heterozygous apoE3/4 frontal cortex homogenates, under non-reduced (NR) and reduced (R) conditions. Western blotting was performed using goat anti-apoE polyclonal antibody. The human brain samples (AD n = 8) are identified according to the Case # code given in Table 1.CC BY · PubMed Central
Figure 6 ApoE3 dimers are present in SK-N-SH cell lysate and rabbit frontal cortex. SK-N-SH cell lysate was analysed under non-reduced (NR) and reduced (R) SDS-PAGE conditions and apoE was detected using anti-apoE C-terminal monoclonal antibody (A). TBS-soluble rabbit brain homogenate was analysed under NR and R conditions and apoE detected using goat anti-apoE polyclonal antibody (B). Two bands marked with asterisks are believed to be due to non-specific cross-reaction with the proteins indicated (asterisks) by Ponceau staining (B).
Figure 6 ApoE3 dimers are present in SK-N-SH cell lysate and rabbit frontal cortex. SK-N-SH cell lysate was analysed under non-reduced (NR) and reduced (R) SDS-PAGE conditions and apoE was detected using anti-apoE C-terminal monoclonal antibody (A). TBS-soluble rabbit brain homogenate was analysed under NR and R conditions and apoE detected using goat anti-apoE polyclonal antibody (B). Two bands marked with asterisks are believed to be due to non-specific cross-reaction with the proteins indicated (asterisks) by Ponceau staining (B).CC BY · PubMed Central
Figure 7 ApoE dimer is not artificially formed during sample homogenisation and processing. Freshly prepared rabbit and frozen human brain tissue (AD apoE3/3, AD 5) was homogenised in a detergent-rich lysis buffer either with or without the thiol-trapping agent iodoacetamide (Iodo) used at a final concentration of 100 mM. The effect of Iodo on the presence of apoE dimers was assessed under non-reduced (NR) and reduced (R) conditions by western blot, using goat anti-apoE polyclonal antibody (A). The extent of thiol-trapping by Iodo was determined by measuring the total thiol present, using DTNB assay (B). Data in B are means of triplicate readings with S.E. represented by error bars. *** P < 0.0001. The human brain sample (AD n = 1) is identified according to the Case # code given in Table 1.
Figure 7 ApoE dimer is not artificially formed during sample homogenisation and processing. Freshly prepared rabbit and frozen human brain tissue (AD apoE3/3, AD 5) was homogenised in a detergent-rich lysis buffer either with or without the thiol-trapping agent iodoacetamide (Iodo) used at a final concentration of 100 mM. The effect of Iodo on the presence of apoE dimers was assessed under non-reduced (NR) and reduced (R) conditions by western blot, using goat anti-apoE polyclonal antibody (A). The extent of thiol-trapping by Iodo was determined by measuring the total thiol present, using DTNB assay (B). Data in B are means of triplicate readings with S.E. represented by error bars. *** P < 0.0001. The human brain sample (AD n = 1) is identified according to the Case # code given in Table 1.CC BY · PubMed Central

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Authors
Elliott, D. A., Halliday, G. M., & Garner, B.
Venue
BMC Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2010-02-20
Volume
11
Issue
1
ISSN
1471-2202
DOI
10.1186/1471-2202-11-23
PMC
PMC2837047
Cited by
31
References
9
Language
en

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