Ferritin confers protection against iron-mediated neurotoxicity and ferroptosis through iron chelating mechanisms in MPP⁺-induced MES23.5 dopaminergic cells

Highlights

• Astrocytes increased ferritin release to respond to iron overload.

• Astrocytes protected MES23.5 dopaminergic cells against MPP ⁺-induced neurotoxicity.

• Exogenous Apoferritin or Ferritin protected against MPP ⁺-induced cell damage.

Abstract

Ferritin is the main iron storage protein and plays an important role in maintaining iron homeostasis. In a previous study, we reported that apoferritin exerted a neuroprotective effect against MPTP by regulation of brain iron metabolism and ferroptosis. However, the precise cellular mechanisms of extracellular ferritin underlying this protection are not fully elucidated. Ferritin was reported to be localized in different intracellular compartments, cytoplasm or released outside cells. Here we demonstrated that the intracellular iron increased after iron treatment in primary cultured astrocytes. These iron-loaded astrocytes released more ferritin in order to buffer extracellular iron. Using co-culture system of primary cultured astrocytes and MES23.5 dopaminergic cells, we showed that ferritin released by astrocytes could enter MES23.5 dopaminergic cells. And primary cultured astrocytes protected MES23.5 dopaminergic cells against 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced neurotoxicity and ferroptosis. In addition, we found that exogenous Apoferritin or Ferritin pretreatment could significantly inhibit MPP⁺-induced cell damage by restoring the cell viability and mitochondrial transmembrane potential (ΔΨm). Furthermore, exogenous Apoferritin and Ferritin might also protect MES23.5 dopaminergic cells against MPP⁺ by decreasing reactive oxygen species (ROS) and inhibiting the increase of the labile iron pool (LIP). This suggests that astrocytes increased ferritin release to respond to iron overload, which might inhibit iron-mediated oxidative damage and ferroptosis of dopamine (DA) neurons in Parkinson's disease (PD).

Introduction

Parkinson's disease (PD) is the second largest neurodegenerative disease in the world [1]. The clinical features of PD are mainly manifested as motor symptoms, including resting tremor, bradykinesia, muscle rigidity, posture and gait dysfunction [2,3]. The loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) is the major pathological feature of PD [4]. At present, there is no effective treatment to prevent or delay the degeneration of DA neurons in the patients of PD. Although the etiology of PD is not fully elucidated, more and more evidence shows that iron-induced oxidative stress is one of the key factors contributing to the pathogenesis of PD [5,6].

Ferritin is a kind of iron storage protein and plays a central role in the regulation of iron metabolism [7,8]. It consists of 24 subunits including heavy-chain ferritin (H-Ferritin) and light-chain ferritin (L-Ferritin) [9]. H-Ferritin has ferroxidase activity, which converts soluble ferrous iron into storable ferric iron [10]. Then ferric iron enters the cavity of ferritin and forms iron core under the action of L-Ferritin [11]. In the cavity of the protein shell, ferritin can bind different amounts of Fe³⁺. In physiological state, ferritin can store nearly 2000 Fe³⁺. While when the cavity is completely saturated, ferritin can store up to 4500 Fe³⁺ [12]. Previous studies showed that there was a large amount of iron deposition in the SNpc of PD patients, while the levels of ferritin in the whole brain and substantia nigra (SN) of PD patients decreased, suggesting the load of ferritin in the SN increased [13]. This might contribute to the increased vulnerability of DA neurons to oxidative stress damage in the SN. Therefore, regulating ferritin levels might protect DA neurons against iron-induced cell damage.

Ferritin was reported to be localized in different intracellular compartments, cytoplasm or released outside cells. This indicates that ferritin might not only be an intracellular iron storage protein, but might be also involved in the iron regulation by extracellular ferritin [14,15]. However, the function of extracellular ferritin in the brain of PD is not fully elucidated. Astrocytes are the most numerous, widely distributed glial cells in the brain. They play an important role in maintaining iron homeostasis and protect neurons in the brain from iron-mediated oxidative stress damage [16]. In a proteomics study on secreted proteins of astrocytes, ferritin was found to be one of the secreted proteins, suggesting that astrocytes might be an important source of secreted ferritin in the brain [17].

Therefore, in our study, we used 1-methyl-4-phenylpyridinium ion (MPP⁺) to establish a PD cell model in MES23.5 dopaminergic cells. The primary cultured astrocytes and MES23.5 dopaminergic cells co-culture system was used to observe the effect of ferritin released by astrocytes on MPP⁺-induced neurotoxicity. We also used exogenous Apoferritin or Ferritin to investigate their effect in MPP⁺-induced PD cell model. Our results demonstrated that primary cultured astrocytes could release ferritin. And the release of ferritin in primary cultured astrocytes increased after ferric ammonium citrate (FAC) treatment. Co-culture experiments of primary cultured astrocytes and MES23.5 dopaminergic cells showed that ferritin released by astrocytes could enter MES23.5 dopaminergic cells and play a protective role for MES23.5 dopaminergic cells against MPP⁺-induced neurotoxicity. We further demonstrated that exogenous Apoferritin or Ferritin could protect MES23.5 dopaminergic cells against MPP⁺ by restoring the cell viability, ΔΨm, and decreasing reactive oxygen species (ROS). Furthermore, Apoferritin and Ferritin might also suppress MPP⁺ or MPP⁺+FAC-induced increase of the labile iron pool (LIP) by binding with intracellular iron, thereby protecting MES23.5 dopaminergic cells.