Study on the Protective Effect of ERP57 on ALS-related Mutant TDP-43 in Neuronal Cells

Introduction

Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disease affecting motor neurons. Almost all ALS cases (97%) and about 50% of frontotemporal dementia (FTD) cases show a pathological form of Tar-DNA binding protein-43 (TDP-43), indicating that TDP-43 plays an important role in neurodegenerative diseases. Previous studies have found that endoplasmic reticulum protein 57 (ERP57), a member of the Protein Disulphide Isomerase (PDI) family, has a protective effect in ALS-related mutant superoxide dismutase 1 (SOD1) mouse models. However, it remains unclear whether ERP57 has a protective effect on the specific pathological TDP-43 in ALS. The aim of this study is to investigate the role of ERP57 in the TDP-43 pathological process and evaluate its potential as a therapeutic target.

Research Source

This paper was completed in collaboration with Sonam Parakh, Emma R. Perri, Marta Vidal, and other colleagues from the Macquarie University Motor Neuron Disease Research Centre, published in “Neuromolecular Medicine”. The paper was received on November 16, 2023, and accepted on April 9, 2024. Professor Julie D. Atkin is the principal investigator of this research.

Research Process

Research Subjects and Methods

Cell Lines:

The study used Mouse neuro-2a neuroblastoma cell line (sourced from CellBank Australia), cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS), at 37°C with 5% CO_2.

Construction and Transfection:

A pCDNA3.1(+) plasmid containing ERP57 was provided by Dr Neil Bulleid from the University of Glasgow. Wild-type TDP-43 and mutant TDP-43m337v with Turbo-GFP tags (from pCMV6-AC-GFP) were also used. Transfection experiments were performed using Lipofectamine-2000 (Invitrogen) according to the manufacturer’s protocol.

Immunocytochemistry and Microscopy:

Neuronal cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.1% Triton-X in PBS. They were then stained with various primary and secondary antibodies, and finally stained with Hoechst 33342 for nuclei. Cells were observed and data recorded using fluorescence microscopy.

Main Research Steps

1. Problem Definition and Hypothesis:

The goal was to determine if ERP57 could have a protective effect against ALS-related mutant TDP-43, preventing its abnormal localization and aggregation in neuronal cells.

2. Cell Line Experiments:

Cell lines with TDP-43 and ERP57 tags were constructed, and cells were observed through immunocytochemistry and fluorescence microscopy. The main observations included TDP-43 localization, inclusion formation, ER stress response, and cell apoptosis.

Data Analysis and Results

Reduced TDP-43 Steady-State Levels:

The study first observed whether ERP57 could have a protective effect on TDP-43 pathology in neuronal cells. Through confocal fluorescence microscopy observation of cells after 72h transfection, it was found that ERP57 and TDP-43 were co-expressed in 99% of transfected cells. Western blotting showed that the steady-state levels of both wild-type and mutant TDP-43 decreased in the presence of ERP57. This was particularly significant at lower expression levels (*p < 0.05).

ERP57 Prevents Abnormal Localization of TDP-43:

Next, the localization of TDP-43 in cells when co-expressed with ERP57 and TDP-43m337v was observed. The results showed that ERP57 could significantly reduce the abnormal localization of TDP-43 in the cytoplasm (*p < 0.05), indicating that ERP57 can prevent the mislocalization of mutant TDP-43.

ERP57 Inhibits Inclusion Formation of Mutant TDP-43:

Quantitative analysis through confocal fluorescence microscopy showed that ERP57 co-expression significantly reduced inclusion formation in cells with mutant TDP-43m337v. Further 3D image analysis showed that ERP57 co-expression could significantly reduce the size of inclusions, indicating that ERP57 played a role in preventing TDP-43 misfolding.

ERP57 Reduces ER Stress and Cell Apoptosis:

Immunocytochemistry showed that ERP57 could significantly reduce ER stress (CHOP immunoreactivity) and cell apoptosis (fragmented or condensed nuclei) induced by mutant TDP-43.

Conclusions and Key Findings

The main findings of this study indicate that:

1. ERP57 can significantly reduce the abnormal localization of mutant TDP-43 in the cytoplasm and reduce the formation and size of inclusions.
2. ERP57 can reduce ER stress and cell apoptosis caused by mutant TDP-43, showing its protective effect.
3. ERP57 may act as a molecular chaperone in the folding pathway, helping to maintain the correct folding and function of TDP-43.

This study demonstrates for the first time the protective effect of ERP57 in ALS-related TDP-43 pathology, further emphasizing the important role of PDI family members in neurodegenerative diseases. ERP57, as a potential therapeutic target, may provide new insights for the treatment of ALS, FTD, and other related neurodegenerative diseases.

Significance and Future Research Directions

This study provides a new perspective for understanding the protective mechanism of ERP57 in ALS and its potential therapeutic applications. Future research could further explore the role of ERP57 in in vivo ALS models and determine specific molecular mechanisms and protein interaction networks. The study also suggests examining the applicability of ERP57 in other neurodegenerative diseases, exploring its potential in broader clinical applications.