Ethanol impairs microtubule formation via interactions at a microtubule associated protein-sensitive site.
Prolonged ethanol abuse has been associated with brain injury caused by impaired synaptogenesis, cellular migration, neurogenesis, and cell signaling, all of which require proper microtubule functioning. However, the means by which ethanol may impair microtubule formation or function and the role that microtubule-associated proteins (MAPs) have in mediating such effects are not clear. In the present studies, purified MAP-deficient (2 mg/mL) and MAP-rich (pre-conjugated; 1 mg/mL) bovine α/β tubulin dimer was allowed to polymerize at 37 °C, forming microtubules in the presence or absence of ethanol (25-500 mM). Microtubule formation was assessed in a 96-well format using a turbidity assay, with absorption measured at 340 nm for 45 min. Additional studies co-exposed α/β tubulin dimers to 50 mM ethanol and purified MAPs (0.1 mg/mL) for 45 min. Polymerization of MAP-deficient tubulin was significantly decreased (at 15-45 min of polymerization) during exposure to ethanol (>25 mM). In contrast, ethanol exposure did not alter polymerization of α/β tubulin dimers pre-conjugated to MAPs, at any concentration. Concurrent exposure of MAP-deficient tubulin with purified MAPs and ethanol resulted in significant and time-dependent decreases in tubulin polymerization, with recovery from inhibition at later time points. The present results suggest that ethanol disrupts MAP-independent microtubule formation and MAP-dependent microtubule formation via direct actions at an MAP-sensitive microtubule residue, indicating that disruption of neuronal microtubule formation and function may contribute to the neurodegenerative effects of binge-like ethanol intake.
Department of Psychology, Tusculum College, 60 Shiloh Road, Greenville, TN 37743, USA.
Katherine J
KJ
Smith
Tracy R
TR
Butler
Mark A
MA
Prendergast
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D000431:Ethanol / Q000494:pharmacology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D014404:Tubulin / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; D014404:Tubulin; D000431:Ethanol
ANOVA; Alcoholism; CNS; G-PEM; MAP-2; MAPS; Neuronal injury; Polymerization; Tubulin; analysis of variance; cAMP; central nervous system; cyclic adenosine monophosphate; microtubule-associated protein 2; microtubule-associated proteins; piperazine-N,N′-bis(2-ethanesulfonic acid) sequisodium salt