Tetramethylpyrazine Nitrone Promotes α-Synuclein Clearance: NRF2-Mediated UPS Activation

Background

Parkinson’s Disease (PD) is a common neurodegenerative disorder primarily characterized by the degeneration of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies, with α-synuclein (α-syn) aggregation as the main component. These pathological features are observed in both familial and sporadic forms of PD. Although the detailed molecular mechanisms of neuronal loss in PD are not yet fully understood, multiple lines of evidence support the crucial role of α-syn in PD pathogenesis.

Current PD treatment strategies mainly include dopamine replacement therapy, which only alleviates motor symptoms without modifying disease progression. Given the key role of α-syn in PD pathogenesis, research focused on α-syn has become particularly important, with strategies such as α-syn clearance, inhibition of its aggregation, and reduction of its expression levels being explored in drug development.

NRF2 (Nuclear Factor Erythroid 2-Related Factor 2) is the main transcriptional regulator of cellular antioxidant stress responses. NRF2 regulates the expression of various antioxidant enzymes and phase II enzyme genes by binding to the Antioxidant Response Element (ARE). Studies have shown that NRF2 can accelerate α-syn clearance by shortening its half-life. Additionally, the absence of NRF2 coexisting with α-syn promotes protein aggregation, neuroinflammation, and neuronal death.

PGC-1α (Peroxisome Proliferator-Activated Receptor γ Co-Activator 1α) is an important transcriptional co-activator that binds to NRF2 to control the expression of mitochondria-related proteins, thereby regulating mitochondrial biogenesis, respiration, and antioxidant defense systems. Research has found that PGC-1α is downregulated in PD brains, and activation or overexpression of PGC-1α can reduce α-syn aggregation.

In this context, this paper proposes to regulate the UPS (Ubiquitin–Proteasome System) to clear α-syn in PD by activating the PGC-1α and NRF2 signaling pathways.

Research Source and Author Information

This paper was jointly authored by Baojian Guo, Chengyou Zheng, Jie Cao, Xiaoling Qiu, Fangcheng Luo, Haitao Li, Simon Mingyuan Lee, Xifei Yang, Gaoxiao Zhang, Yewei Sun, Zaijun Zhang, and Yuqiang Wang. The research team is from institutions including the School of Pharmacy at University of Jinan, Peking University Shenzhen Graduate School, Zunyi Medical University, University of Macau, and Shenzhen Center for Disease Control and Prevention. The paper was published in Volume 26 of “Neuromolecular Medicine” in 2024.

Research Workflow

Research Subjects and Sample Processing

This study used SH-SY5Y and PC12 cell models stably expressing human A53T mutant α-syn, as well as transgenic mouse models (HA53T) expressing the same A53T mutant α-syn. Various subjects were pre-treated and underwent different experiments to test the effects of TBN (Tetramethylpyrazine Nitrone), including protein extraction, ELISA measurements, Western blotting, immunofluorescence staining, proteasome activity assays, and molecular docking studies.

Behavioral Tests

To assess motor function in mice, pole tests and open field tests were conducted. In the experiments, HA53T transgenic mice were divided into different groups, receiving either TBN or saline treatment, followed by behavioral tests to evaluate the neuroprotective effect of TBN.

Protein Extraction and Determination

Through protein extraction from cell and mouse samples, the study determined α-syn content and its phosphorylated forms, and observed the effects of TBN on the expression and activity of related proteins such as PGC-1α and NRF2.

Proteasome Activity Assay

The study used the Proteasome-Glo™ kit to measure the enzymatic activities of three subunits in the proteasome, including trypsin-like, chymotrypsin-like, and caspase-like activities, to explore the in vitro effects of TBN on UPS.

Main Research Findings

Neuroprotective Effects of TBN

In the HA53T mouse model, after TBN treatment, there was no significant change in mouse body weight, but their motor coordination (pole test) significantly improved, activity distance increased, while serum α-syn levels decreased, and the production of oxidative damage products 3-NT and 4-HNE was reduced.

TBN Promotes α-Syn Clearance

In vitro results showed that TBN significantly reduced MPP+ and 6-OHDA-induced α-syn overexpression and decreased the expression of phosphorylated α-syn (Ser 129). Under conditions of mutant α-syn overexpression in PC12 cells, TBN significantly increased caspase-like activity without affecting trypsin-like and chymotrypsin-like activities. On the other hand, mg132 (proteasome inhibitor) and CQ (lysosome inhibitor) significantly blocked the α-syn clearance effect of TBN, indicating that TBN accelerates α-syn degradation through both UPS and ALP pathways. Further studies showed that TBN could significantly upregulate PGC-1α and NRF2 expression, and the effect of TBN on α-syn clearance was completely blocked under NRF2 siRNA action.

Research Conclusions and Significance

The study demonstrates that TBN has significant neuroprotective effects on various PD models. TBN enhances UPS degradation of α-syn by activating the PGC-1α/NRF2 pathway and alleviates oxidative stress and mitochondrial dysfunction caused by mutant α-syn, thus providing a new avenue for potential PD disease-modifying therapies. This research not only reveals the potential of TBN in PD treatment but also emphasizes the importance of PGC-1α/NRF2 activation as a key target in the fundamental mechanisms of PD.

Research Highlights

• Discovery and validation of TBN’s neuroprotective effects on multiple PD models: This small molecule can significantly reduce α-syn-induced oxidative damage and promote its clearance.
• In-depth exploration of TBN’s mechanism of action: Demonstrated NRF2 as a key transduction factor, regulating UPS activity to clear α-syn through the PGC-1α/NRF2 pathway.
• Proposal of a potential disease-modifying therapy: Compared to α-syn antibodies, TBN showed advantages in crossing the BBB and activating endogenous cellular defense, laying the foundation for future clinical applications.

This study not only provides a scientific basis for TBN but also brings new hope to the field of PD treatment. Future research will further validate the specific molecular targets and mechanisms of action of TBN, thus promoting its application in clinical practice to benefit more patients.