In vitro analysis of the purified VASH1-SVBP complex, the most abundant tubulin carboxypeptidase in the brain, revealed that the presence of L-Dopa-tubulin alters the complex's binding to microtubules and reduces its carboxypeptidase activity. These findings suggest that L-Dopa incorporation into tubulin modifies microtubule properties and their interaction with this enzyme.

To confirm the involvement of L-Dopa-microtubules in the observed dendritic spine alterations in wild-type neurons, we examined the effect of L-Dopa treatment also affect neurons lacking the enzymes of the α-tubulin detyrosination/tyrosination cycle. In these neurons, L-Dopa cannot be incorporated into α-tubulin due to the absence of the ligase (in TTL knockout neurons) or reduced levels of the substrate: detyrosinated α-tubulin (in SVBP knockout neurons). L-Dopa treatment did not alter dendritic spine density or excitatory synapses in TTL KO or SVBP KO neurons, clearly indicating that the post-synaptic defects observed in WT neurons are due to L-Dopa incorporation into tubulin. Further analysis revealed that in WT neurons, L-Dopa altered microtubule dynamics in the dendritic shaft by increasing catastrophe frequency and reducing comet lifetime, resulting in fewer microtubules entering dendritic spines and decreased spine resistance to pruning.