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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.

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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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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.
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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.

Mentions: To address the possibility that the observed apoE dimerisation may occur during tissue homogenization and processing for electrophoresis, freshly prepared rabbit brain and frozen human frontal cortex tissue (AD apoE3/3) were homogenized in buffer containing the thiol trapping agent iodoacetamide. Homogenization was also performed using a detergent-rich lysis buffer to delipidate apoE-containing lipoproteins and help prevent the possibility of dimers forming spontaneously on lipoprotein particles during homogenization and processing. The presence of 100 mM iodoacetamide did not result in a decrease in apoE homodimer levels in either rabbit or human brain tissue (Fig 7A). The thiol-trapping efficiency of 100 mM iodoacetamide was confirmed by spectrophotometric analysis of the total thiol concentration of the homogenates using Ellman's reagent (5,5'-Dithio-bis(2-nitrobenzoic acid)). The total thiol concentration was significantly reduced in both rabbit and human brain homogenates (84% and 96%, respectively) treated with iodoacetamide (Fig 7B). This data demonstrates that apoE dimers are not artificially formed during tissue homogenization and processing, and further indicates that apoE dimers are generated in the brain.


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

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

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.
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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.
Mentions: To address the possibility that the observed apoE dimerisation may occur during tissue homogenization and processing for electrophoresis, freshly prepared rabbit brain and frozen human frontal cortex tissue (AD apoE3/3) were homogenized in buffer containing the thiol trapping agent iodoacetamide. Homogenization was also performed using a detergent-rich lysis buffer to delipidate apoE-containing lipoproteins and help prevent the possibility of dimers forming spontaneously on lipoprotein particles during homogenization and processing. The presence of 100 mM iodoacetamide did not result in a decrease in apoE homodimer levels in either rabbit or human brain tissue (Fig 7A). The thiol-trapping efficiency of 100 mM iodoacetamide was confirmed by spectrophotometric analysis of the total thiol concentration of the homogenates using Ellman's reagent (5,5'-Dithio-bis(2-nitrobenzoic acid)). The total thiol concentration was significantly reduced in both rabbit and human brain homogenates (84% and 96%, respectively) treated with iodoacetamide (Fig 7B). This data demonstrates that apoE dimers are not artificially formed during tissue homogenization and processing, and further indicates that apoE dimers are generated in the brain.

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