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Updated: Aug 29, 2025
Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis
Published on: July 16, 2021
Random forest modelling demonstrates microglial and protein misfolding features to be key phenotypic markers in
Olivia M Rifai1,2,3,4,5, James Longden2, Judi O'Shaughnessy2,3,4
1Translational Neuroscience PhD Programme, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK.
Abstract:
Clinical heterogeneity observed across patients with amyotrophic lateral sclerosis (ALS) is a known complicating factor in identifying potential therapeutics, even within cohorts with the same mutation, such as C9orf72 hexanucleotide repeat expansions (HREs). Thus, further understanding of pathways underlying this heterogeneity is essential for appropriate ALS trial stratification and the meaningful assessment of clinical outcomes. It has been shown that both inflammation and protein misfolding can influence ALS pathogenesis, such as the manifestation or severity of motor or cognitive symptoms. However, there has yet to be a systematic and quantitative assessment of immunohistochemical markers to interrogate the potential relevance of these pathways in an unbiased manner. To investigate this, we extensively characterised features of commonly used glial activation and protein misfolding stains in thousands of images of post-mortem tissue from a heterogeneous cohort of deeply clinically profiled patients with a C9orf72 HRE. Using a random forest model, we show that microglial staining features are the most accurate classifiers of disease status in our panel and that clinicopathological relationships exist between microglial activation status, TDP-43 pathology, and language dysfunction. Furthermore, we detected spatially resolved changes in fused in sarcoma (FUS) staining, suggesting that liquid-liquid phase shift of this aggregation-prone RNA-binding protein may be important in ALS caused by a C9orf72 HRE. Interestingly, no one feature alone significantly impacted the predictiveness of the model, indicating that the collective examination of all features, or a combination of several features, is what allows the model to be predictive. Our findings provide further support to the hypothesis of dysfunctional immune regulation and proteostasis in the pathogenesis of C9-ALS and provide a framework for digital analysis of commonly used neuropathological stains as a tool to enrich our understanding of clinicopathological relationships within and between cohorts. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Insights
Microglial staining patterns accurately classify disease status in amyotrophic lateral sclerosis (ALS) patients with C9orf72 expansions. These findings highlight immune dysregulation and protein misfolding in C9-ALS pathogenesis.
Area of Science:
- Neuroscience
- Immunology
- Pathology
Background:
- Clinical heterogeneity in amyotrophic lateral sclerosis (ALS) complicates therapeutic development, even in patients with the C9orf72 hexanucleotide repeat expansion (HRE).
- Understanding pathways like inflammation and protein misfolding is crucial for ALS trial stratification and outcome assessment.
- A systematic, quantitative assessment of immunohistochemical markers for these pathways is lacking.
Purpose of the Study:
- To systematically and quantitatively assess glial activation and protein misfolding markers in C9orf72-HRE ALS patient post-mortem tissue.
- To investigate clinicopathological relationships between these markers and clinical phenotypes.
- To establish a framework for digital analysis of neuropathological stains in ALS research.
Main Methods:
- Utilized thousands of images from post-mortem brain tissue of clinically profiled C9orf72-HRE ALS patients.
- Applied immunohistochemical staining for glial activation and protein misfolding markers.
- Employed a random forest model for quantitative analysis and classification of disease status.
Main Results:
- Microglial staining features were the most accurate classifiers of disease status in the studied panel.
- Clinicopathological relationships were identified between microglial activation, TDP-43 pathology, and language dysfunction.
- Spatially resolved changes in FUS staining suggested a role for liquid-liquid phase separation in C9orf72-HRE ALS.
Conclusions:
- Microglial activation status and protein misfolding pathways are implicated in C9orf72-HRE ALS pathogenesis.
- Digital analysis of neuropathological stains provides a framework for understanding clinicopathological relationships.
- Combined assessment of multiple features, rather than single markers, enhances predictive power for ALS subtypes.
