Detection of Seeding Activity of Human Superoxide Dismutase 1 Aggregates in Postmortem Neural Tissues of Familial and Sporadic Amyotrophic Lateral Sclerosis Patients

Background Introduction

Amyotrophic Lateral Sclerosis (ALS) is a rapidly progressing neurodegenerative disease with an average survival time of 2 to 5 years after diagnosis. Major symptoms of ALS include muscle twitching, muscle fatigue, cramps, and weakness. In the late stages, symptoms include weight loss, speech loss, and paralysis, with respiratory failure being the main cause of death. The etiology of ALS is complex, with approximately 90% of ALS cases being sporadic (sporadic ALS, sALS), and the remaining 10% being hereditary familial (familial ALS, fALS) cases. Previous studies have shown the presence of aggregated metalloprotein human superoxide dismutase 1 (Superoxide Dismutase 1, SOD1) in the neural tissues of ALS patients, especially prominent in familial ALS patients associated with SOD1 gene mutations. Additionally, SOD1 aggregates have been detected in other forms of ALS, including the most common familial ALS associated with abnormal expansions in the chromosome 9 open reading frame 72 (C9ORF72) gene and sporadic ALS.

Existing studies suggest that SOD1 aggregates may possess prion-like seeding and propagation properties. There is a need to develop new diagnostic methods to gain a deeper understanding of the dynamics of SOD1 in ALS pathology and to evaluate its potential application as an ALS biomarker. In this context, this study aims to develop a seed amplification detection method based on real-time quaking-induced conversion (RT-QuIC) technology to measure the seeding activity of SOD1 in postmortem spinal cord and motor cortex tissues from ALS patients.

Source of the Paper and Author Information

This study was co-authored by Justin K. Mielke, Mikael Klingeborn, Eric P. Schultz, Erin L. Markham, Emily D. Reese, Parvez Alam, Ian R. Mackenzie, Cindy V. Ly, Byron Caughey, and Neil R. Cashman. The research institutions involved include McLaughlin Research Institute, University of Montana, Rocky Mountain Laboratories, University of British Columbia, and Washington University. The paper was published in Volume 147, Page 100 of the 2024 edition of Acta Neuropathologica. The manuscript was received on February 1, 2024, revised on June 6, 2024, and formally accepted for publication on June 7, 2024.

Research Methods

Workflow

The study’s workflow includes the following key steps:

1. Preparation and Purification of Human SOD1 Plasmid: The research team constructed and expressed a plasmid containing the full-length human SOD1 gene, expressed and purified it using E. coli strain BL21 (DE3), and confirmed the quality of the expressed SOD1 using purification techniques such as metal ion affinity chromatography.

2. Preparation and Processing of Human Postmortem Spinal Cord and Motor Cortex Tissues: The research team extracted tissues including cervical and thoracic spinal cord and motor cortex from ALS patients and healthy controls, and prepared tissue homogenates with different concentration gradients.

3. Immunocapture Experiments: Using specific anti-SOD1 antibody-conjugated magnetic beads, the aggregates of SOD1 were captured to control the specificity of the RT-QuIC detection.

4. SOD1 RT-QuIC Detection: The research developed a novel SOD1 RT-QuIC detection method using an optimized reaction system, including key reaction buffer components (e.g., SOD1 substrate, GuHCl, sodium acetate, and ThT), conducting the detection in microplates, and measuring the aggregation reaction process using fluorescence intensity.

5. Transmission Electron Microscopy Analysis: To confirm that the aggregates produced by RT-QuIC are amyloid fibrils, the research team observed the morphology of the aggregates using transmission electron microscopy.

6. Data Analysis and Statistics: By fitting RT-QuIC curves to calculate fluorescence enhancement values and lag phases, the differences between various types of ALS cases were analyzed.

Samples and Experimental Subjects

1. Thoracic spinal cords and motor cortex provided by the National Institutes of Health (NIH) for sporadic ALS and disease controls.
2. Familial ALS spinal cord tissue samples provided by the National Brain Bank (Georgetown Brain Bank).
3. Neurological control samples from the teams of Ian Mackenzie and Cindy Ly at UBC and others at Washington University.
4. Data obtained from multiple laboratories, ensuring the reliability and accuracy of the data through numerous repeated and control experiments.

Research Results

Detection Characteristics of SOD1 Substrate

Using the SOD1 RT-QuIC reaction system, the study found that SOD1 aggregates could significantly increase fluorescence intensity, especially in tissues from ALS patients, while this phenomenon was absent in healthy controls. This indicates that these SOD1 aggregates have self-replicating and propagating properties.

RT-QuIC Detection Results of Different ALS Types

1. Familial ALS (SOD1 Mutation): RT-QuIC detected a large amount of SOD1 aggregates in the spinal cord and motor cortex tissues of these patients, showing a shorter lag phase and higher fluorescence intensity.
2. Familial ALS (C9ORF72 Mutation): Corresponding SOD1 aggregates were detected, showing propagation characteristics similar to those of SOD1 mutation patients.
3. Sporadic ALS: SOD1 aggregates were also detected in the tissues of sporadic ALS patients, indicating that wild-type SOD1 in these patients also possesses prion-like self-propagation ability.

SOD1 Seeding Activity in Different Anatomical Regions

Comparing tissues from different anatomical regions (such as thoracic spinal cord and motor cortex) of ALS patients, the study showed that the SOD1 seeding activity in the motor cortex was significantly higher than in the spinal cord, suggesting that the rate of neurodegeneration in the ALS pathological process may be closely related to the anatomical location.

Conclusions and Significance

This study developed and validated an RT-QuIC-based SOD1 seeding activity detection method, capable of accurately detecting SOD1 aggregates in the neural tissues of different types of ALS patients. This method demonstrates potential as an ALS biomarker, particularly significant for the diagnosis and research of sporadic ALS patients. With further optimization and application, this detection method may provide new tools for early diagnosis and efficacy evaluation of ALS.

Research Highlights

1. Development of a New Method: The study developed the first RT-QuIC-based ALS-related SOD1 seeding activity detection method, with high sensitivity and specificity.
2. Detection of Multiple ALS Types: The method was validated for application in various ALS pathological types, providing new evidence for the pathological mechanisms of different ALS types.
3. Anatomical Specificity Differences: Significant differences in SOD1 seeding activity were found in the tissues of ALS patients from different anatomical regions, providing a new perspective for the study of ALS pathological expansion mechanisms.

Future Prospects

Based on this study, the next step will be to explore the potential application of this detection method in bio-samples obtainable from living patients and to continue researching the commonalities and differences between different ALS types, thereby better understanding the pathogenesis of this complex disease and developing targeted therapeutic strategies.