Alcohol-related brain damage in humans

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.

Figure 1. Histological examination of prefrontal cortex neuronal cells from controls and alcoholics.

Cortical pyramidal cells (upper two panels), and subcortical neurons (lower two panels) of control and alcoholic tissue sections were visualised by light microscopy. White bar represents 50 μm for 10x, 10 μm for 50x, and 5 μm for 100x lenses. Examples of the differences in the cytoplasm surrounding the nuclei of cells from alcoholic or control tissue sections have been marked with white arrows.

Figure 2. Profiling of cytosolic proteins from the prefrontal cortex of control and alcoholic subjects.

Control (C) or alcoholic (A) prefrontal cortex cytosolic proteins were resolved by 1D PAGE and proteins stained with colloidal Coomassie. Alcoholic subjects displayed a prominent reduction in the level of protein staining at protein bands of ∼270 kDa and ∼50 kDa (upper panel). Protein levels were visualized by Western blotting (lower panel). Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PIMT, protein-L-isoaspartate O-methyltransferase.

Figure 3. 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.

Figure 4. Western blot of α- and β-tubulins in nuclear and membrane fractions from the prefrontal cortex of control and alcoholic subjects.

Control (C) or alcoholic (A) prefrontal cortex nuclear and membrane fractions were resolved by 1D PAGE and proteins Western blotted for α- and β-tubulins.

Figure 5. Profiling of membrane-fraction proteins from the prefrontal cortex of control and alcoholic subjects.

Control (C) or alcoholic (A) prefrontal cortex membrane fraction proteins were resolved by 1D PAGE and proteins stained with colloidal Coomassie. Alcoholic subjects displayed a prominent reduction in the level of protein staining at a protein band of ∼270 kDa and upregulation at ∼112 kDa (upper panel). Protein levels of transmembranous (∼112 kDa) Na⁺,K⁺-ATPase α-subunit, and oligomeric forms of the α-subunit were visualized by Western blotting (middle panel). The level of transmembranous α-subunit was quantified using actin for normalisation (lower panel). For marked significance: *** = p<0.001.

Figure 6. Profiling of cytosolic proteins from the caudate nucleus, hippocampus and cerebellum of control and alcoholic subjects.

Control (C) or alcoholic (A) cytosolic proteins from caudate nucleus, hippocampus, and cerebellum were resolved by 1D PAGE and proteins stained with colloidal Coomassie (upper panel). The positions of the ∼50 kDa α- and β-tubulin protein bands are marked with arrows. Cytosolic proteins were Western blotted for α- and β-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (middle panel), and protein level changes quantified using GAPDH for normalization (lower panel). For marked significance: * = p<0.05, ** = p<0.01, *** = p<0.001.