L-Dopa incorporation into tubulin permanently alters the detyrosination/tyrosination cycle, microtubules dynamics and neuronal differentiation

Congreso; Congreso SAIB; 2022

SAIB

Microtubules are key elements of the neuronal cytoskeleton. Differential expression of tubulin isotypes and a variety of post-translational modifications constitute the code that modulates microtubule properties and functions. The reversible removal of the tyrosine residue from the C-terminal end of α-tubulin involves the enzymes tubulin tyrosine ligase and the detyrosinase complexes, composed of a vasohibin (VASH) and a small vasohibin-binding protein (SVBP), and the recently described MATCAP. Previously, we demonstrated that L-3,4-dihydroxyphenylalanine (L-Dopa) is incorporated, in vitro and in living cells, into α-tubulin at the same position as tyrosine . We found, using soluble brain extracts, that L-Dopa was not released by the endogenous carboxypeptidase, under the conditions that allow rapid release of tyrosine. We also reported that L-Dopa treatment impairs mitochondrial axonal transport and reduces the affinity of the motor KIF5B for L-Dopa-microtubules. Now, we have analyzed the interaction between the VASH-SVBP complex and microtubules enriched in L-Dopa-tubulin using single-molecule TIRF. We found a reduction in the complex binding to L-Dopa-microtubules and in its carboxypeptidase activity. In cultured mouse hippocampal neurons, we examined the effect of L-Dopa on neuronal differentiation and dendritic spine density. We observed a delay in the establishment of polarity and transition to stage III in WT neurons, but not in neurons without carboxypeptidase activity, and a reduction in the number of mature dendritic spines. Based on our results, we hypothesize that L-Dopa incorporation into tubulin and microtubules alters the detyrosination/tyrosination cycle and modifies microtubules with a direct implication in neuronal differentiation and synapse formation.