July 2023
Qian Zheng, Pengfei Ma, Pan Yang, Suzhen Zhai, Meina He, Xiangming Zhang, Qiuxia Tu, Ling Jiao, Lan Ye, Zhanhui Feng, Chunlin Zhang

 

Highlights

• Alpha lipoic acid (ALA) ameliorates MPTP-induced motor deficits in mice.

• ALA decreases iron levels by upregulating FTH1 and downregulating DMT1.

• ALA prevents ferroptosis effectively by inhibiting the downregulation of glutathione peroxidase 4 (GPX4) and cysteine/glutamate transporter (xCT) in PD.

• Mechanistic study indicates that the activation of SIRT1/NRF2 pathway is involved the upregulation effect of GPX4 and FTH1.


Abstract

Parkinson’s disease (PD) is a neurodegenerative disease. Ferroptosis shares several features with PD pathophysiology, and anti-ferroptosis molecules are neuroprotective in PD animal models. As an antioxidant and iron chelating agent, alpha lipoic acid (ALA) has a neuroprotective effect on PD; however, the influence of ALA on ferroptosis in PD remains unclear. This study aimed to determine the mechanism of ALA in regulating ferroptosis in PD models. Results showed that ALA could ameliorate motor deficits in PD models and regulate iron metabolism by upregulating ferroportin (FPN) and ferritin heavy chain 1 (FTH1) and downregulating iron importer divalent metal transporter 1 (DMT1). Moreover, ALA decreased the accumulation of reactive oxygen species (ROS) and lipid peroxidation, rescued mitochondrial damage, and prevented ferroptosis effectively by inhibiting the downregulation of glutathione peroxidase 4 (GPX4) and cysteine/glutamate transporter (xCT) in PD. Mechanistic study indicated that the activation of SIRT1/NRF2 pathway was involved in the upregulation effect of GPX4 and FTH1. Thus, ALA ameliorates motor deficits in PD models by regulating iron metabolism and mitigating ferroptosis through the SIRT1/NRF2 signaling pathway.

 

Introduction

Parkinson’s disease (PD) is a common neurodegenerative disease. The four motor symptoms of PD include bradykinesia, postural instability, rigidity, and tremor, which are accompanied by the loss of dopaminergic (DA) neurons [1]. The search for agents with disease-modifying effects for PD is still pending. Despite symptomatic treatment options available for PD, patients still experience a decline in independence due to disease progression. Therefore, developing a treatment that targets the pathological mechanism of PD is imperative.

Iron is an essential cofactor for metabolic processes, but excessive iron content can generate reactive oxygen species (ROS) and trigger cell death. In PD, the deposition of iron in the substantia nigra (SN) of the brain is associated with neuronal loss [2]. Cellular iron accumulation in PD may be related to the imbalance of iron metabolism in the brain [3], suggesting that an imbalance in iron homeostasis may cause neuronal death in PD due to impaired iron metabolism in the brain. Neurons take up iron from transferrin via divalent metal transporter 1 (DMT1) and transferrin receptor 1 (TfR1) and export excess iron via ferroportin (FPN) [3]. The upregulation of DMT1 may cause iron accumulation and the degeneration of DA neurons in PD [4]. A study has shown reduced FPN expression in PD animal models [5]. Therefore, restoring brain iron homeostasis may be a potential therapeutic strategy for PD.

Ferroptosis is a new kind of regulated cell death that is initiated by abnormal iron metabolism and severe lipid peroxidation [6]. PD and ferroptosis exhibit highly similar pathological changes, such as lipid peroxidation, iron deposition, and antioxidant system deficiency [6]. Furthermore, the inhibition of ferroptosis could alleviate motor behavioral and tyrosine hydroxylase (TH) neuronal loss in PD models [7]. Ferroptosis is regulated by iron metabolism, which includes iron uptake, storage, and export. Therefore, targeting ferroptosis is a crucial treatment strategy for managing PD [7].

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) plays a crucial role in cellular antioxidant defense mechanisms in PD [8]. It transcriptionally regulates the expression of ferroptosis-related factors, including the cystine/glutamate transporter system xC-/xCT and glutathione peroxidase 4 (GPX4) [9]. In addition, NRF2 pathway could regulate the expression of iron metabolism-related proteins, such as DMT1, FPN, and FTH1, which influence the intracellular iron homeostasis [10]. The regulation of NRF2 signaling is a promising strategy to modulate the progression of PD [11].

Silence information regulator 1 (SIRT1) is an NAD+-dependent protein deacetylase that is involved in regulating numerous cellular processes. Its enzymatic activity is observed to decrease in patients with PD, impairing its ability to resist neuronal damage [12]. Furthermore, SIRT1 is an upstream target of NRF2 that can enhance resistance against oxidative stress. Therefore, activation of the SIRT1/NRF2 signaling pathway is a potential target for PD treatment.

Alpha-Lipoic acid (ALA) is an enzyme cofactor with iron chelator and antioxidant properties and exerts protective effects against PD [13]. It facilitated the clearance of iron accumulation by regulating DMT1 expression in PD models [13]. Whether ALA can regulate FPN and FTH1 and the mechanism by which ALA alleviates ferroptosis in PD remain unclear. In PD models, ALA significantly upregulated SIRT1 [14] and NRF2 [15]to prevent oxidative stress. Whether ALA could alleviate ferroptosis in PD by activating the SIRT1/NRF2 signaling pathway is also unclear. Therefore, this study aimed to identify the specific mechanism of ALA in regulating ferroptosis in PD models.

Section snippets

Experimental design of mice

In the first experiment, 45 mice were divided randomly into control, PD, and ALA groups (15 for each group) (Fig. 3A). In the second experiment, 30 mice were divided into control, PD, ALA, EX527, and ML385 groups (six for each group) (Fig. 7A). The PD, ALA, EX527, and ML385 groups were intraperitoneally injected with MPTP (30 mg/kg/d) for 7 consecutive days (from day 3 to day 10). The ALA group was intraperitoneally injected with ALA (50 mg/kg/day) from day 1 to day 14. The EX527 (5 mg/kg/day)

ALA shows neuroprotection in PD cell model

Cell viability was expressed as an MTT conversion rate. The results showed that different concentrations of ALA could ameliorate the 6-OHDA-induced damage in PC12 cells, and 10 µM of ALA was the greatest protective effect (Figure S1).

ALA decreases lipid peroxidation and iron levels in PD cell model

One of the main mechanisms of ferroptosis is lipid peroxidation, and many ROS are the central product of lipid peroxidation. Hence, the effect of ALA on lipid peroxidation levels were detected. Flow cytometry and fluorescence results showed that ROS was increased

Discussion

PD is a neurodegenerative disorder that is currently incurable and characterized by various symptoms. Iron deposition is among the key factors in the etiology of PD, and inhibiting this process and ferroptosis may help prevent the progression of the disease [13]. However, the underlying mechanism for this effect is still unclear.

PD is caused by the loss of DA neurons in SN, and restoring DA neurodegeneration is an effective method for PD treatment. In this study, pretreatment with ALA restored

Conclusion

This study demonstrated that ALA could ameliorate motor deficits in PD models by regulating iron metabolism and mitigating ferroptosis. The possible underlying molecular mechanisms involve FTH1-mediated iron metabolism and GPX4-mediated ferroptosis via the SIRT1/NRF2 pathway (Fig. 9). Additionally, activation of the SIRT1/NRF2 pathway may be a valid target for PD treatment.

Ethics statement

The animal study was reviewed and approved by Ethical Committee of the Guizhou Medical University (No. 2000958).

Funding

This work was supported by National Natural Science Foundation of China (No. 81860248; No.82160234); the Science and Technology Project of Guizhou Province, China (No. ZK[2021]113); the Characteristic field project in the Education Department of Guizhou Province, China (No. KY[2021]066); the Youth Science and Technology Talent Development Project in the Education Department of Guizhou Province, China (No.KY[2022]225); the Science and Technology Fund of Guizhou Health Commission

Credit authorship contribution statement

Conceptualization: QZ, CZ, ZF; Data curation: QZ and PM; Formal analysis: QZ and PM; Funding acquisition: QZ, LJ,CZ and ZF; Investigation: CZ and ZF; Methodology:QZ, PM, PY, XZ, LJ, LY, SZ, MH and QT; Project administration: CZ and ZF; Resources:QZ, LJ,CZ and ZF; Software:QZ, PM, PY, XZ, LY, SZ, MH and QT; Supervision: QZ, CZ, ZF; Validation: CZ and ZF; Visualization: QZ, PM; Writing-original draft: QZ; Writing-review and editing: CZ and ZF.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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