Effects of Clemizole on Parkinson’s Disease Pathology Induced by α-Synuclein Fibril Formation

Background Introduction

Parkinson’s Disease (PD) is a typical neurodegenerative disease, mainly associated with mitochondrial dysfunction and oxidative stress. However, current therapeutic approaches targeting these pathological events have not successfully translated from laboratory to clinical application. One important reason for this failure in translation is the use of traditional models that cannot truly reproduce the disease pathology and progression. Therefore, to address this issue, this study adopted a more physiologically relevant model by exposing α-synuclein pre-formed fibrils (PFF) to SH-SY5Y cells and Sprague Dawley rats to explore the role of Transient Receptor Potential Canonical 5 (TRPC5) in PD-like pathology and its potential therapeutic effects through alleviating oxidative stress and improving mitochondrial health.

Paper Source

This research paper was conducted by Bhupesh Vaidya, Pankaj Gupta, Soumojit Biswas, Joydev K. Laha, Ipsita Roy, Shyam Sunder Sharma, and others at the National Institute of Pharmaceutical Education and Research in India, published in the journal “Neuromolecular Medicine” in 2024.

Research Process and Methods

Research Subjects

The research subjects included SH-SY5Y cells and Sprague Dawley rats. The study mainly used in vivo and in vitro models, establishing PD-like pathology through injection and cell treatment methods.

Purification and Synthesis of α-Synuclein

The study first used expression vectors to transform E. coli BL21(DE3) cells to obtain recombinant human α-synuclein. Through a series of purification steps, including ion exchange chromatography, purified α-synuclein monomers were finally prepared, which were then used to synthesize PFF in buffer solution. The quality was verified by methods such as Thioflavin T (THT) fluorescence detection and transmission electron microscopy.

In Vivo Experimental Design

α-Synuclein PFF was injected into specific striatal locations of Sprague Dawley rats to induce PD-like pathology. Rats were divided into multiple groups, receiving different doses of Clemizole treatment (10 mg/kg and 30 mg/kg) for 14 days. The rats were subsequently dissected, and various molecular and biochemical parameters were measured.

Behavioral Tests

Rotarod test, Y-maze spontaneous alternation test, and passive avoidance test were conducted to assess the rats’ motor coordination, working memory, and fear-conditioned response. The data from these tests were used to compare differences between different treatment groups.

Biochemical and Molecular Analysis

Methods such as immunoblotting, real-time quantitative PCR, and calmodulin activity assay were used to detect the expression of proteins and genes including α-synuclein, p-α-synuclein, TRPC5, TH, PGC-1α, TFAM in striatal and midbrain samples. Additionally, the activity of mitochondrial complex I and the expression level of ND2 gene were measured.

Cell Culture and In Vitro Experiments

SH-SY5Y cells were treated with α-synuclein PFF as well as Clemizole or TRPC5 siRNA, and cell viability tests, oxidative stress analysis, mitochondrial function detection, and immunocytochemical staining were performed.

Data Analysis

All experimental data were analyzed using GraphPad Prism 8 software, employing one-way ANOVA analysis and Tukey’s test, with p < 0.05 considered as the level of statistical significance.

Main Findings

Reduction in α-Synuclein Expression

Clemizole significantly reduced the expression of α-synuclein and p-α-synuclein in both midbrain and striatum, thereby alleviating neurobehavioral abnormalities in PFF-treated rats. This result was also applicable in cell experiments, where Clemizole showed a dose-dependent effect.

Decrease in TRPC5 Expression

TRPC5 expression was significantly increased in PFF-treated rats and cell models, while Clemizole effectively reduced this expression, suggesting a potential role of TRPC5 in PD pathology.

Behavioral Improvement

In PFF-treated rats, Clemizole significantly improved their performance in behavioral tests, restoring motor coordination and memory capacity, while increasing TH expression levels.

Mitochondrial Function Improvement

Clemizole significantly increased the expression of mitochondrial biogenesis-related genes PGC-1α and TFAM, enhanced the activity of mitochondrial complex I and ND2 gene levels, thereby improving overall mitochondrial health.

Oxidative Stress and Calcium Signaling Pathway

Clemizole significantly reduced total ROS and mitochondrial ROS levels in PFF-treated SH-SY5Y cells, restoring cell membrane potential. Additionally, changes in the expression of calcium signaling pathway-related genes Calmodulin and Parvalbumin were also restored.

Conclusion and Significance

This study is the first to use a human α-synuclein PFF model system to screen pharmacological agents, which has important theoretical and practical value for PD pathology. The results show that Clemizole significantly improved PD-like pathology induced by α-synuclein PFF, including oxidative stress levels, mitochondrial function, and behavioral abnormalities, by inhibiting the TRPC5 channel. This provides potential targets and methods for developing new PD therapeutic drugs and offers new insights for research on other neurodegenerative diseases.

Research Highlights

The highlight of this study is the first use of a human α-synuclein PFF model system for drug screening, demonstrating the multiple improvement effects of Clemizole on PD pathology, especially its significant therapeutic effects in TRPC5 channel regulation, mitochondrial function restoration, and oxidative stress reduction.

The above research lays the foundation for further exploration of the potential benefits of TRPC5 channels in other neurodegenerative diseases, while also providing strong support for the development of new drugs.