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, V; DITAMO, Y; ARCE, CA; BISIG, CG

Cordoba

Congreso; XXXIII Congreso anual de la Sociedad Argentina de Investigacion en Neurociencias (SAN); 2018

Sociedad Argentina de Investigación en Neurociencias

The C-terminal tyrosine (Tyr) of the α-tubulin chain is subjected to post-translational removal andre-addition in a process termed the ?detyrosination/tyrosination cycle?. We showed in previous studiesusing soluble rat brain extracts that L-3,4-dihydroxyphenylalanine (L-Dopa) is incorporated into the samesite as 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.Dopa-tubulin had microtubule-forming capability similar to that of Tyr- and Glu-tubulin. CAD cells (aneuron-like cell line derived from mouse brain) are easily cultured under differentiating andnon-differentiating conditions, and can be treated with L-Dopa. Treatment of CAD cells with L-Doparesulted in reduction of microtubule dynamics in neurites, most likely due to L-Dopa incorporation intoα-tubulin. To study the functional effects of this alteration we treated primary culture rat hippocampalneurons with L-Dopa. We observed a reduction in the number of mitochondria and mitochondrial trafficalong axonal microtubules. This findings may be due to an abnormal interaction between microtubulesand molecular motors which participate in organelles transport. We hypothesized that this alterationscould be associated with movement disorders sometimes observed in patients treated with L- Dopa forprolonged periods.