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Apolipoprotein-E forms dimers in human frontal cortex and hippocampus.

Elliott DA, Halliday GM, Garner B - BMC Neurosci (2010)

Bottom Line: 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 level of dimerisation was not significantly different when control and AD samples were compared.Similar apoE3 dimers were also detected in lysates of SK-N-SH neuroblastoma cells and in freshly prepared rabbit brain homogenates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Prince of Wales Medical Research Institute, Randwick NSW 2031, Australia.

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, approximately 12% of apoE was present as a homodimer and approximately 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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Related in: MedlinePlus

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).
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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).

Mentions: Analysis of SK-N-SH cell lysates under non-reducing conditions revealed the presence of the ~95 kDa apoE homodimer and a more prominent (than human brain) ~43 kDa heterodimer (Fig 6A). Since the ~43 kDa dimer could theoretically represent a disulphide-linked apoE N-terminal domain homodimer (a predicted MW of ~44 kDa), we used a C-terminal specific monoclonal antibody in this experiment and the detection of the ~43 kDa band indicates an intact C-terminus. This rules out the presence of disulphide-linked N-terminal domain homodimer. The ~43 kDa band may represent an apoE3-apoA-II heterodimer as has been previously described in human plasma and CSF [30,33].


Apolipoprotein-E forms dimers in human frontal cortex and hippocampus.

Elliott DA, Halliday GM, Garner B - BMC Neurosci (2010)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2837047&req=5

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).
Mentions: Analysis of SK-N-SH cell lysates under non-reducing conditions revealed the presence of the ~95 kDa apoE homodimer and a more prominent (than human brain) ~43 kDa heterodimer (Fig 6A). Since the ~43 kDa dimer could theoretically represent a disulphide-linked apoE N-terminal domain homodimer (a predicted MW of ~44 kDa), we used a C-terminal specific monoclonal antibody in this experiment and the detection of the ~43 kDa band indicates an intact C-terminus. This rules out the presence of disulphide-linked N-terminal domain homodimer. The ~43 kDa band may represent an apoE3-apoA-II heterodimer as has been previously described in human plasma and CSF [30,33].

Bottom Line: 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 level of dimerisation was not significantly different when control and AD samples were compared.Similar apoE3 dimers were also detected in lysates of SK-N-SH neuroblastoma cells and in freshly prepared rabbit brain homogenates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Prince of Wales Medical Research Institute, Randwick NSW 2031, Australia.

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, approximately 12% of apoE was present as a homodimer and approximately 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.

Show MeSH
Related in: MedlinePlus