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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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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.
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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.

Mentions: We also used an additional rigorous extraction protocol employing extraction buffer that contained the detergent Triton-X100. This was done in order to maximise recovery of apoE that may be associated with TBS-insoluble material. Both the control and AD samples were found to contain apoE homodimers when samples were extracted in detergent-containing buffer (Fig 3). These data are very similar to the results obtained with the TBS-soluble homogenates (Fig 1).


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

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

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

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

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.
Mentions: We also used an additional rigorous extraction protocol employing extraction buffer that contained the detergent Triton-X100. This was done in order to maximise recovery of apoE that may be associated with TBS-insoluble material. Both the control and AD samples were found to contain apoE homodimers when samples were extracted in detergent-containing buffer (Fig 3). These data are very similar to the results obtained with the TBS-soluble homogenates (Fig 1).

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