L-Dopa incorporation into tubulin affects microtubules dynamics, neuro differentiation and dendritic spine density

ZORGNIOTTI A; RAMIREZ-RIOS S; SANYAL C; DITAMO Y; MOUTIN MJ; PERIS L; BISIG CG

Congreso; IUBMB-EMBO FOCUSED MEETING ON EMERGING CONCEPTS OF THE NEURONAL CYTOSKELETON (6TH EDITION); 2023

EMBO

Microtubules are key elements of the eukaryotic cytoskeleton composed of α- and β-tubulin heterodimers. Differential expression of tubulin isotypes and a variety of post-translational modifications constitutes the tubulin code that modulates the properties and function of microtubules (1). One of the first described tubulin post-translational modifications is the cyclic removal and re-addition of the tyrosine (Tyr) residue encoded at the C-terminal end of the α-subunit, know as detyrosination/tyrosination cycle (2,3). This modification of α-tubulin involves different enzymes: the tubulin tyrosine ligase (TTL) and the detyrosinase complexes composed of a vasohibin (VASH1 or VASH2) and a small vasohibin-binding protein (SVBP) (4,5). Recently, MATCAP, a third tubulin carboxypeptidase, has been described (6). The role of this post-translational modification in cell physiological processes remains poorly understood; nevertheless, there is increasing evidence of its importance in specialized microtubule functions (1,7).In previous studies, we have demonstrated that L-3,4-dihydroxyphenylalanine (L-Dopa) is incorporated into α-tubulin at the same position as Tyr, both in vitro and in living cells. However, using soluble brain extracts, we found that L-Dopa was not released by the endogenous carboxypeptidase under the conditions that allow rapid release of Tyr from tubulin. In addition, we reported that L-Dopa treatment impairs mitochondrial transport along the axon and that the molecular motor KIF5B shows a reduced affinity for L-Dopa-microtubules. (8) Now, we analyzed the interaction between the VASH-SVBP complex and microtubules enriched in L-Dopa-tubulin in vitro, using single-molecule TIRF and immunofluorescence assays. We found a reduction in both the capacity of the complex to bind L-Dopa-microtubules and its carboxypeptidase activity, supporting the idea of L-Dopa irreversible incorporation into tubulin. Additionally, we examined in mouse hippocampal neurons the effect of L-Dopa treatment on neuronal differentiation in vitro. We observed a delay in the establishment of polarity and transition to stage III in WT neurons, but not in neurons without carboxypeptidase activity (SVBP KO). Based on our results, we hypothesize that the irreversible incorporation of L-Dopa into tubulin and microtubules permanently alters the detyrosination/tyrosination cycle and modifies microtubules dynamics with a direct implication in neuronal differentiation.