Cholesterol and 24S-OHC Induce Neuronal Apoptosis and Necroptosis, but Not Ferroptosis Despite Elevated Iron Levels

Selective loss of dopaminergic neurons in the substantia nigra (SN) is the main pathological feature of Parkinson's disease (PD), and nigral iron deposition is a key factor in the pathogenesis of PD. Excessive iron can lead to increased production of Reactive Oxygen Species (ROS) in cells and cause cell damage. The abnormal synthesis and metabolism of Cholesterol in the brain are closely related to the occurrence of central nervous system diseases, but the mechanisms remain unclear. This experiment investigates the effects and mechanisms of Cholesterol and 24S-hydroxycholesterol (24S-OHC) on SH-SY5Y neurons and primary cultured ventral mesencephalon neurons. The viability of SH-SY5Y neurons and primary cultured ventral mesencephalon (VM) neurons was detected by cell counting kit-8 (CCK-8) assay. The intracellular ROS levels were detected by flow cytometry. The expressions of proteins related to Ferroptosis, Apoptosis, and Necroptosis were detected by western blot. The mRNA expression of pro-inflammatory cytokines was detected by Real-Time PCR. In neurons treated with Cholesterol or 24S-OHC, the protein expressions of iron regulatory protein 1 (IRP1), Transferrin Receptor 1 (TfR1), divalent metal transporter 1 (DMT1), and ferritin were increased, while the expression of Ferroportin 1 (FPN1) was decreased, thus leading to increased intracellular iron levels. However, Glutathione Peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11; also called x-CT), Ferroptosis suppressor protein 1 (FSP1), and malondialdehyde (MDA), the indicators of Ferroptosis, showed no obvious alterations. Cholesterol induced the increased expressions of pro-inflammatory cytokines interleukin-1 beta (IL-1β) and IL-6 in neurons, and the Erk-NF-κB pathway changed accordingly. Moreover, the expressions of cleaved Caspase-3, the ratio of Bax/Bcl-2, receptor-interacting protein kinase 1 (RIPK1), RIPK3, and phosphorylated Mixed Lineage Kinase domain-like (p-MLKL) in neurons were also elevated by 24S-OHC treatment. Morphologically, we found nuclear membrane breakdown, chromatin condensation, organelle expansion, cytoplasmic vacuolization, and contents outflow in neurons, which were further signs of neuronal Apoptosis and Necroptosis. Additionally, co-treatment with deferoxamine (DFO) can partially reduce cleaved Caspase-3 expression but cannot reduce p-MLKL expression. Our findings suggest that both Cholesterol and 24S-OHC can raise the level of Reactive Oxygen Species (ROS) and activate IRP1 to increase iron influx and decrease iron outflow in neurons. Despite the increase in iron content, Ferroptosis is not observed. Cholesterol also increases the expressions of IL-1β and IL-6, which may be related to the Erk-NF-κB pathway, while 24S-OHC can cause both Apoptosis and Necroptosis. DFO can only partially reduce Apoptosis but cannot prevent Necroptosis in neurons.

24-Hydroxy-cholesterol; Apoptosis; Cholesterol; Iron metabolism; Necroptosis.