ABSTRACT Previous studies have shown that L-Dopa, a tyrosine analog used in Parkinson’s disease treatment, can be incorporated into α-tubulin C-terminal tail via the tubulin tyrosine ligase (TTL) and polymerize into microtubules. In this work, we demonstrated that mature wild type hippocampal neurons treated with L-Dopa exhibited reduced dendritic spine density, primarily affecting mature dendritic spines. In these neurons, L-Dopa treatment significantly reduced tyrosinated α-tubulin levels without altering detyrosinated or Δ2 α-tubulin levels, suggesting the formation of a new tubulin pool, likely composed of L-Dopa-α-tubulin. In vitro analysis of the activity of the purified VASH1-SVBP complex, the most abundant tubulin carboxypeptidase in brain, revealed that L-Dopa incorporation into α-tubulin modified the binding of the complex to microtubules and reduced its carboxypeptidase activity. These results suggest that L-Dopa incorporation into tubulin alters the properties of microtubules and affects their ability to interact with the enzyme. To confirm the implication of L-Dopa-microtubules in dendritic spine alterations observed in wild type neurons, we analyzed the effect of L-Dopa treatment in neurons lacking the enzymes of the α-tubulin detyrosination/tyrosination cycle. In these cells, L-Dopa cannot be incorporated into α-tubulin due to the absence of the ligase (in TTL KO neurons) or the reduction of detyrosinated α-tubulin levels (in SVBP KO neurons). L-Dopa treatment did not modify dendritic spine density in TLL KO or SVBP KO neurons, clearly demonstrating that the alterations in dendritic spines seen in WT neurons are due to the incorporation of L-Dopa into tubulin. Further analysis revealed that L-Dopa treatment decreased the percentage of spines containing excitatory synapses in wild type neurons, but not in TTL KO or SVBP KO neurons, suggesting a cumulative synaptic defect due to L-Dopa incorporation into microtubules. Additionally, L-Dopa altered microtubule dynamics by increasing catastrophe frequency and reducing comet lifetime, which led to fewer microtubules entering dendritic spines and decreased spine resistance to pruning. Taken together, our results demonstrate that L-Dopa incorporation into α-tubulin drastically affects synaptic homeostasis, reaffirming the importance of balanced detyrosination/tyrosination of tubulin within the synaptic compartment. The abnormal dynamics of L-Dopa-microtubules and the reduction of dendritic spines and excitatory synapses highlight a novel mechanism of L-Dopa-induced synaptotoxicity.

L-Dopa incorporation into tubulin alters microtubule dynamics and reduces dendritic spine invasion and synapse maintenance