PROTEOMIC INSIGHTS INTO THE NEUROPROTECTIVE EFFECTS OF <EM>EUCALYPTUS CITRIODORA</EM> ESSENTIAL OIL IN A 6-HYDROXYDOPAMINE-INDUCED N2A CELL MODEL

Parkinson’s disease (PD) is a progressive neurodegenerative disorder for which current therapies are largely symptomatic and fail to halt disease progression. Natural compounds with multitarget bioactivities offer promising neuroprotective strategies. Here, we assessed the neuroprotective effect of Eucalyptus citriodora essential oil (ECEO) in a 6-hydroxydopamine (6-OHDA)–induced murine neuroblastoma cell (N2a) model. ECEO pretreatment preserved cell viability (as indicated by FDA assay) and reduced membrane damage (as evidenced by lower LDH release) compared with 6-OHDA exposure. Consistently, intracellular reactive oxygen (ROS) and nitric oxide (NO) were significantly reduced in ECEO-pretreated cells, indicating effective attenuation of stress markers. To explore the underlying molecular mechanisms, we performed differential proteomic profiling by 2D electrophoresis followed by mass spectrometry and functional analysis. 6-OHDA induced pronounced alterations in proteins linked to proteostasis, metabolism, and cellular stress response, whereas ECEO pretreatment shifted the proteomic signature toward control-like profiles. ECEO modulated Key components of protein quality control and mitochondrial function, including the endoplasmic reticulum chaperone HSPA5 (BIP/GRP78), the ATPase VCP, the proteasome subunits (PSMB4 and PSMA3), the mitochondrial scaffold proteins PHB1, and the complex I subunit NDUFS3, suggesting reinforcement of proteostasis and mitochondrial networks. Enrichment analyses highlighted coordinated regulation of biological processes involved in carbon metabolism, mitochondrial protein degradation, and aerobic respiration. Overall, these findings suggest that ECEO mitigates 6-OHDA-induced toxicity in N2a cells by reducing oxidative stress and stabilizing proteostasis and mitochondrial networks, laying the groundwork for further translational studies.