POST-TRANSLATIONAL INCORPORATION OF L-DOPA INTO THE C-TERMINUS OF α-TUBULIN IN LIVING CELLS AFFECTS MICROTUBULE DYNAMICS AND MITOCHONDRIAL TRAFFIC.

ZORGNIOTTI A; FILIBERTI, F; DITAMO, Y; ARCE, CA; BISIG, CG

Congreso; Congreso Sociedad Argentina de Neurociencias; 2018

The C-terminal tyrosine (Tyr) of the α-tubulin chain is subjected to post-translational removal and readditionin a process termed the ?detyrosination/tyrosination cycle?. We showed in previous studies usingsoluble rat brain extracts that L-3,4-dihydroxyphenylalanine (L-Dopa) is incorporated into the same siteas Tyr. We now demonstrate that L-Dopa incorporation into tubulin also occurs in living cells. Wedetected such incorporation by determining the ?tyrosination state? of tubulin before and after incubationof cells in the presence of L-Dopa. The presence of a tubulin isospecies following L-Dopa incubation thatwas not recognized by antibodies specific to Tyr- and Glu-tubulin was presumed to reflect formation ofDopa-tubulin. L-Dopa was identified by HPLC as the C-terminal compound bound to α-tubulin. Dopatubulinhad microtubule-forming capability similar to that of Tyr- and Glu-tubulin. CAD cells (a neuronlikecell line derived from mouse brain) are easily cultured under differentiating and non-differentiatingconditions, and can be treated with L-Dopa. Treatment of CAD cells with L-Dopa resulted in reduction ofmicrotubule dynamics in neurites, most likely due to L-Dopa incorporation into α-tubulin. To study thefunctional effects of this alteration we treated primary culture rat hippocampal neurons with L-Dopa. Weobserved a reduction in the number of mitochondria and mitochondrial traffic along axonal microtubules.This findings may be due to an abnormal interaction between microtubules and molecular motors whichparticipate in organelles transport. We hypothesized that this alterations could be associated withmovement disorders sometimes observed in patients treated with L- Dopa for prolonged periods.