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The Loss of α- And β-Tubulin Proteins Are a Pathological Hallmark of Chronic Alcohol Consumption and Natural Brain Ageing

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The Loss of α- And β-Tubulin Proteins Are a Pathological Hallmark of Chronic Alcohol Consumption and Natural Brain Ageing

Wajana L Labisso et al. Brain Sci.

Abstract

Repetitive excessive alcohol intoxication leads to neuronal damage and brain shrinkage. We examined cytoskeletal protein expression in human post-mortem tissue from Brodmann's area 9 of the prefrontal cortex (PFC). Brain samples from 44 individuals were divided into equal groups of 11 control, 11 alcoholic, 11 non-alcoholic suicides, and 11 suicide alcoholics matched for age, sex, and post-mortem delay. Tissue from alcoholic cohorts displayed significantly reduced expression of α- and β-tubulins, and increased levels of acetylated α-tubulin. Protein levels of histone deacetylase-6 (HDAC6), and the microtubule-associated proteins MAP-2 and MAP-tau were reduced in alcoholic cohorts, although for MAPs this was not significant. Tubulin gene expressions increased in alcoholic cohorts but not significantly. Brains from rats administered alcohol for 4 weeks also displayed significantly reduced tubulin protein levels and increased α-tubulin acetylation. PFC tissue from control subjects had reduced tubulin protein expression that was most notable from the sixth to the eighth decade of life. Collectively, loss of neuronal tubulin proteins are a hallmark of both chronic alcohol consumption and natural brain ageing. The reduction of cytosolic tubulin proteins could contribute to the brain volumetric losses reported for alcoholic patients and the elderly.

Keywords: HDAC6; MAP-2; MAP-tau; acetylation; ageing; alcohol-related brain damage; alcoholism; pre-frontal cortex; α-tubulin; β-tubulin.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Resolution of human PFC proteins from cytosolic, nuclear, and membrane-enriched fractions from control (C), alcoholic (A), suicide (S), and suicide alcoholic (SA) patients. Prefrontal cortex proteins were resolved by one dimensional polyacrylamide gel electrophoresis and then stained with colloidal Coomassie. Reduced protein staining at ≈50 kDa (marked with arrowheads) was solely in the cytosolic protein fractions of alcoholic and suicide alcoholic patients.
Figure 2
Figure 2
Protein levels of PFC proteins visualized by Western blotting. (A) Cytosolic proteins resolved by one dimensional polyacrylamide gel electrophoresis (1D PAGE) were Western blotted using antibodies to HDAC6, β-tubulin, α-tubulin, acetylated α-tubulin, and GAPDH. Blotting panels from six sets of matched control (C), alcoholic (A), suicide (S), and suicide alcoholic (SA) patients are included. (B) Cytosolic proteins resolved by 1D PAGE were Western blotted with antibodies to MAP-2, MAP-tau, and GAPDH. Blotting panels from three sample sets of matched control (C), alcoholic (A), suicide (S), and suicide alcoholic (SA) patients are included.
Figure 3
Figure 3
Quantitation of Western blots. Western blots of proteins from control (C), alcoholic (A), suicide (S), and suicide alcoholic (SA) patients were quantified using densitometry, with GAPDH levels used for blot normalization. Histograms are representative of means ± standard deviation, n = 9–11. For marked significance: * = p < 0.05, ** = p < 0.01. Control values were set at 100%.
Figure 4
Figure 4
Quantitation of α- and β-tubulin gene expression by qRT-PCR. Relative gene expression of α-tubulin and β-tubulin in control (C), alcoholic (A), suicide (S), and suicide alcoholic (SA) patients was quantified using qRT-PCR. Expression of the genes beta actin, beta-2-microglobulin, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta were used as housekeeping genes. Histograms are representative of means ± SDs, n = 6. Control values were set at 1.
Figure 5
Figure 5
Profiling of brain cytoplasmic proteins from control (C) and ethanol-fed (E) rats, and Western blotting. (A) Brain cytosolic proteins from control (C) or ethanol-fed (E) rats resolved by one dimensional polyacrylamide gel electrophoresis were stained with Coomassie (upper panel). Protein staining at ≈50 kDa (marked with an arrowhead) was reduced in ethanol-fed rats. Proteins were Western blotted using a panel of antibodies (middle and lower panels). (B) Western blots were quantified by densitometry, with GAPDH levels used for blot normalization, and data presented as box plots. For marked significance: * = p < 0.05, ** = p < 0.01. Control values were set at 100%.
Figure 6
Figure 6
Resolution of proteins from the PFC of humans of progressive age and Western blotting. (A) PFC cytosolic proteins were resolved by one dimensional polyacrylamide gel electrophoresis and stained with colloidal Coomassie. A reduction of the protein band at ≈50 kDa was apparent for most subjects over 60 years of age (marked with arrowheads). Western blotting confirmed that the reduced protein staining reflected a lowering of α- and β-tubulin expression. (B) Quantification of the changes of protein expression of α- and β-tubulins, and GAPDH across the age range of 21–86 years, with data presented as box plots. GAPDH blots were normalized to total protein staining. For marked significance: * = p < 0.05, ** = p < 0.01. (C) Densitometric quantitation of the ≈50 kDa protein band across the age range of 21–86 years, with data presented as box plots. For marked significance: * = p < 0.05.
Figure 6
Figure 6
Resolution of proteins from the PFC of humans of progressive age and Western blotting. (A) PFC cytosolic proteins were resolved by one dimensional polyacrylamide gel electrophoresis and stained with colloidal Coomassie. A reduction of the protein band at ≈50 kDa was apparent for most subjects over 60 years of age (marked with arrowheads). Western blotting confirmed that the reduced protein staining reflected a lowering of α- and β-tubulin expression. (B) Quantification of the changes of protein expression of α- and β-tubulins, and GAPDH across the age range of 21–86 years, with data presented as box plots. GAPDH blots were normalized to total protein staining. For marked significance: * = p < 0.05, ** = p < 0.01. (C) Densitometric quantitation of the ≈50 kDa protein band across the age range of 21–86 years, with data presented as box plots. For marked significance: * = p < 0.05.

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