An L-DOPA-PLP cyclic adduct exerts oxidative stress on -Synuclein

Levodopa (L-DOPA) is widely used as a therapeutic agent to alleviate motor symptoms in Parkinson disease (PD). L-DOPA is converted into dopamine by the pyridoxal 5′-phosphate (PLP) dependent enzyme L-aromatic amino acid decarboxylase (AADC), responsible for the regulation of this neurotransmitter levels. It is known that L-DOPA bound to PLP via a Schiff base covalent linkage can irreversibly cyclize into an adduct via the Pictet-Spengler condensation reaction in vitro, forming a Pictet-Spengler adduct (PsP), either free in solution or at the AADC active site under certain experimental conditions1,2. In addition, several AADC deficiency pathogenic variants undergo this nonenzymatic reaction, as a consequence of their structural alterations at the active site3 . L-DOPA alone has already been shown to react via this mechanism with acetaldehyde, to form salsolinol, which has been detected in the urine of PD patients4 . It is possible that unmetabolized L-DOPA may react with PLP also in vivo, possibly leading to PLP depletion. Indeed, this depletion could impair PLP-dependent enzymes. Furthermore, PsP itself may interact with ⍺-synuclein (Syn), contributing to neurodegenerative pathology. Here, we investigated the impact of PsP on Syn aggregation and explored the molecular mechanisms underlying this interaction. We observed that PsP caused a dose-dependent inhibition of Syn fibrillation, correlated with the formation of oxidized forms of Syn. The oxidative effect of PsP appears linked to its chemical instability at pH 7.4. Notably, at pH 6 where PsP is stable, neither inhibition of fibrillation nor Syn oxidation were observed. Even though native mass spectrometry revealed that PsP induces immediate protein compaction, isothermal titration calorimetry (ITC) detects no enthalpically driven interaction between the two. This suggests that the observed compaction may result from hydrophobic interactions. In conclusion, while PsP does not engage in specific molecular interactions with Syn, its chemical instability may disrupt the protein oxidative balance, adding to the existing stressors contributing to Syn pathology in PD.