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Alcohol-related brain damage in humans.

Erdozain AM, Morentin B, Bedford L, King E, Tooth D, Brewer C, Wayne D, Johnson L, Gerdes HK, Wigmore P, Callado LF, Carter WG - PLoS ONE (2014)

Bottom Line: BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin β II, and α- and β-tubulins in alcoholics, and these were validated and quantitated by Western blotting.There was also a significant reduction in proteasome activity in alcoholics.One dimensional PAGE of membrane-enriched fractions detected a reduction in β-spectrin protein levels, and a significant increase in transmembranous α3 (catalytic) subunit of the Na+,K+-ATPase in alcoholic subjects.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, United Kingdom; Department of Pharmacology, University of the Basque Country, and Centro de Investigación Biomédica en Red de Salud Mental, Spain.

ABSTRACT
Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann's area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin β II, and α- and β-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in α-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in β-spectrin protein levels, and a significant increase in transmembranous α3 (catalytic) subunit of the Na+,K+-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of α-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic α- and β-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics.

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Related in: MedlinePlus

Quantification of protein level and activity differences between control and alcoholic subjects.Protein levels were quantified by Western blotting, and the levels of isoaspartate protein damage and proteasome activity determined. For quantitation of the ratio of acetylated α-tubulin to total α-tubulin, the figure is representative of those subjects that displayed visible total α-tubulin signal (6 of the 10 subjects assayed). For marked significance: * = p<0.05, ** = p<0.01, *** = p<0.001. Abbreviations: PIMT, protein-L-isoaspartate O-methyltransferase.
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pone-0093586-g003: Quantification of protein level and activity differences between control and alcoholic subjects.Protein levels were quantified by Western blotting, and the levels of isoaspartate protein damage and proteasome activity determined. For quantitation of the ratio of acetylated α-tubulin to total α-tubulin, the figure is representative of those subjects that displayed visible total α-tubulin signal (6 of the 10 subjects assayed). For marked significance: * = p<0.05, ** = p<0.01, *** = p<0.001. Abbreviations: PIMT, protein-L-isoaspartate O-methyltransferase.

Mentions: To validate and quantify this dramatic loss of the cytoskeletal proteins β-spectrin, and α- and β- type tubulins specifically within alcoholic brain tissue, we assessed their protein levels by Western blotting – Figure 2 lower section. Western blot analyses confirmed a significant 36% decrease in spectrin β II (p = 0.0002), significant 56% decrease in α-tubulin (p = 0.0017) and significant 83% decrease in β-tubulin levels (p<0.0001) for the 10 alcoholic subjects – Figure 3. The alcoholic subjects for which the level of either α- or β-tubulins were below the Western blotting detection threshold have been expressed as 100% protein loss. Cytosolic GAPDH levels were stable throughout all control and alcoholic subjects investigated and were used for protein normalisation.


Alcohol-related brain damage in humans.

Erdozain AM, Morentin B, Bedford L, King E, Tooth D, Brewer C, Wayne D, Johnson L, Gerdes HK, Wigmore P, Callado LF, Carter WG - PLoS ONE (2014)

Quantification of protein level and activity differences between control and alcoholic subjects.Protein levels were quantified by Western blotting, and the levels of isoaspartate protein damage and proteasome activity determined. For quantitation of the ratio of acetylated α-tubulin to total α-tubulin, the figure is representative of those subjects that displayed visible total α-tubulin signal (6 of the 10 subjects assayed). For marked significance: * = p<0.05, ** = p<0.01, *** = p<0.001. Abbreviations: PIMT, protein-L-isoaspartate O-methyltransferase.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974765&req=5

pone-0093586-g003: Quantification of protein level and activity differences between control and alcoholic subjects.Protein levels were quantified by Western blotting, and the levels of isoaspartate protein damage and proteasome activity determined. For quantitation of the ratio of acetylated α-tubulin to total α-tubulin, the figure is representative of those subjects that displayed visible total α-tubulin signal (6 of the 10 subjects assayed). For marked significance: * = p<0.05, ** = p<0.01, *** = p<0.001. Abbreviations: PIMT, protein-L-isoaspartate O-methyltransferase.
Mentions: To validate and quantify this dramatic loss of the cytoskeletal proteins β-spectrin, and α- and β- type tubulins specifically within alcoholic brain tissue, we assessed their protein levels by Western blotting – Figure 2 lower section. Western blot analyses confirmed a significant 36% decrease in spectrin β II (p = 0.0002), significant 56% decrease in α-tubulin (p = 0.0017) and significant 83% decrease in β-tubulin levels (p<0.0001) for the 10 alcoholic subjects – Figure 3. The alcoholic subjects for which the level of either α- or β-tubulins were below the Western blotting detection threshold have been expressed as 100% protein loss. Cytosolic GAPDH levels were stable throughout all control and alcoholic subjects investigated and were used for protein normalisation.

Bottom Line: BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin β II, and α- and β-tubulins in alcoholics, and these were validated and quantitated by Western blotting.There was also a significant reduction in proteasome activity in alcoholics.One dimensional PAGE of membrane-enriched fractions detected a reduction in β-spectrin protein levels, and a significant increase in transmembranous α3 (catalytic) subunit of the Na+,K+-ATPase in alcoholic subjects.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, University of Nottingham, Royal Derby Hospital Centre, Derby, United Kingdom; Department of Pharmacology, University of the Basque Country, and Centro de Investigación Biomédica en Red de Salud Mental, Spain.

ABSTRACT
Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann's area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects. BA 9 tissue homogenisation and one dimensional polyacrylamide gel electrophoresis (PAGE) proteomics of cytosolic proteins identified dramatic reductions in the protein levels of spectrin β II, and α- and β-tubulins in alcoholics, and these were validated and quantitated by Western blotting. We detected a significant increase in α-tubulin acetylation in alcoholics, a non-significant increase in isoaspartate protein damage, but a significant increase in protein isoaspartyl methyltransferase protein levels, the enzyme that triggers isoaspartate damage repair in vivo. There was also a significant reduction in proteasome activity in alcoholics. One dimensional PAGE of membrane-enriched fractions detected a reduction in β-spectrin protein levels, and a significant increase in transmembranous α3 (catalytic) subunit of the Na+,K+-ATPase in alcoholic subjects. However, control subjects retained stable oligomeric forms of α-subunit that were diminished in alcoholics. In alcoholics, significant loss of cytosolic α- and β-tubulins were also seen in caudate nucleus, hippocampus and cerebellum, but to different levels, indicative of brain regional susceptibility to alcohol-related damage. Collectively, these protein changes provide a molecular basis for some of the neuronal and behavioural abnormalities attributed to alcoholics.

Show MeSH
Related in: MedlinePlus