Sequential Mitochondrial Transplantation Abrogates Agmatine-Mediated Neuroprotection Against 6-OHDA Toxicity in SH-SY5Y Cells

Objective: Parkinson disease (PD) involves dopaminergic neuron loss from mitochondrial dysfunction and oxidative stress. We investigated the combined, sequential effects of mesenchymal stem cell–derived mitochondrial transplantation (MitoTr) and pharmacological agmatine modulation in a PD–like undifferentiated SH-SY5Y cellular model. Methods: Undifferentiated SH-SY5Y cells were exposed to 6-hydroxydopamine (6-OHDA; 350 μM) for 24 hours and sequentially treated with mitochondria (5 or 10 μg/mL) and agmatine (100 μM; 2 hours post-MitoTr). Cell viability was assessed via MTT assays (n = 8 independent experiments). Total-lysate cytochrome c ELISA (n = 3) and Western blots for DJ-1, REDD1, and α-synuclein (n = 4) were performed across 4 core groups. Results: 6-OHDA significantly reduced viability compared with Control (mean [SEM], 0.50 [0.05] vs 1.00 [0.04]; P < .001); postinjury agmatine restored it to near-control levels (mean [SEM], 0.97 [0.20]; P < .001\) vs 6-OHDA), while agmatine alone had no effect (P > .99). Conversely, MitoTr-containing groups showed significantly lower viability than Control, Agmatine, and Agmatine + 6-OHDA groups (P < .001). Most MitoTr arms fell significantly below 6-OHDA alone (P < .05), except the 5 μg/mL triple group (P = .13). Expression of DJ-1, REDD1, and α-synuclein decreased numerically relative to Control without statistical significance (P > .05). Total-lysate cytochrome c was significantly elevated only in the triple-combination group compared with the Control group (mean [SEM], 36.01 [3.65] versus 22.63 [1.08] ng/mL; P = .03). Conclusions: Postinjury agmatine rescued 6-OHDA-induced viability loss, but sequential MitoTr followed by agmatine caused an additional marked reduction in viability rather than synergistic neuroprotection. Molecular alterations remained exploratory and nonsignificant. This MitoTr-associated loss may reflect antiproliferative effects in neuroblastoma-derived cells, underscoring the need for optimized protocols and nonmalignant models.