C9orf72 Hexanucleotide Repeat Expansions Impair Microglial Response in ALS Patients — In-depth Report on the November 2025 Issue of Nature Neuroscience

1. Academic Background and Research Motivation

Amyotrophic lateral sclerosis (ALS) is a disabling neurodegenerative disease characterized by progressive loss of motor neurons, with most patients dying within three years of disease onset. Beyond motor symptoms, a portion of patients also present cognitive and behavioral impairment. Genetic evidence shows that ALS has strong hereditary susceptibility, with the most common mutation being a GGGGCC hexanucleotide repeat expansion (HRE) at the C9orf72 (chromosome 9 open reading frame 72) gene. This expansion, commonly found in patients with ALS and frontotemporal dementia (FTD), is known to range from 38 to about 1600 repeat units. The pathogenesis of ALS involves not only neurons, but also glial cells such as microglia and astrocytes, which play critical roles in neuroinflammation. However, the specific mechanisms by which C9orf72 HRE influences glial cell function, especially in microglia, remain unclear.

In ALS, neuroinflammation and its cellular mechanisms have become a research focus. Previous studies have shown that C9orf72 is mainly expressed in myeloid cells, especially microglia. Animal experiments indicated that loss of C9orf72 can result in abnormal immune responses, enhanced myeloid cell proliferation, and lysosomal dysfunction. Nevertheless, the specific variation and response mechanisms of human glial cells under these mutations—particularly the differences between genetic and sporadic ALS (sALS)—remain unclear at the forefront of research. To address these scientific pain points, the authors conducted a systematic investigation to reveal the molecular and cellular mechanisms by which C9orf72 HRE affects glial cells in ALS patients, evaluating its impact on the function, transcriptional state, and intercellular communication of both microglia and astrocytes, aiming to provide a theoretical basis for future patient stratification and precision treatment.

2. Paper Source and Author Information

The paper, titled “C9orf72 hexanucleotide repeat expansions impair microglial response in ALS,” was published in the November 2025, Volume 28 issue of Nature Neuroscience. The first authors include Pegah Masrori, Baukje Bijnens, and Laura Fumagalli (all as co-first authors); the corresponding authors are Pegah Masrori, Renzo Mancuso, and Philip van Damme. The authors are mainly affiliated with KU Leuven, University of Antwerp, and other renowned neuroscience research institutes in Europe, showcasing the collaborative strength of multiple institutions.

3. Detailed Interpretation of Research Flow

1. Study Design and Sample Collection

This study adopted a multi-level and systematic design that integrates postmortem human CNS tissues, iPSC-derived cell models, and mouse xenotransplantation models. It enrolled ALS patients (including both C9orf72 HRE and sporadic types, five for each) and healthy controls, obtaining their motor cortex and spinal cord tissues for single-nucleus RNA sequencing (snRNA-seq). Meanwhile, the researchers used human iPSC-derived microglia (including C9orf72 repeat expansion, C9orf72 knockout, and their respective isogenic controls), transplanted them into the brains of immunodeficient mice, and conducted a range of functional and molecular biology experiments.

a) Postmortem Tissue Single-Nucleus Sequencing and Cell Subpopulation Analysis

By snRNA-seq, the researchers systematically identified major cell types and their proportions in motor cortex and spinal cord samples, totaling 156,252 nuclei. The specific cell types included: astrocytes, endothelial cells, neurons, microglia, oligodendrocytes and precursors; natural killer cells were also identified in the spinal cord. For microglia and astrocyte subpopulations, the authors integrated external large datasets (such as the Gerrits et al. study with 132,628 microglial nuclei), using clustering and marker analysis to ultimately identify several microglial subpopulations (such as homeostatic microglia [HM], transitioning microglia [TM], disease-associated microglia [DAM], etc.), as well as six major astrocyte subpopulations.

b) Gene Expression Analysis and Pathway Enrichment

The research mainly focused on C9orf72 expression levels across cell types and found the highest expression in microglia, with significantly reduced expression in C9orf72 HRE carrier microglia, confirming C9orf72 haploinsufficiency (loss-of-function). Based on gene expression changes, the researchers further used weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA) to evaluate the enrichment of key pathways related to lysosomal, phagocytic, inflammatory, and energy metabolic functions.

c) Xenograft Cell Model

To validate findings from postmortem tissues and exclude bystander effects (such as the CNS microenvironment), researchers differentiated iPSCs into microglial precursors, including C9orf72 HRE type, C9orf72 knockout, and their respective isogenic controls, and achieved gene editing via CRISPR-Cas9. These human microglial precursors were then transplanted into the brains of immunodeficient mice (rag2−/− il2g−/− hcsf1ki). After 3 or 6 months, human microglia were isolated by FACS and subjected to single-cell RNA sequencing for cellular state and response analysis.

d) Analysis of Lysosomal and Phagocytic Function in iPSC-derived Microglia

The authors in vitro differentiated human iPSCs into microglia, used immunostaining/high-resolution expansion microscopy to monitor distribution and morphological changes of lysosomal enzymes (such as cathepsin D, CTSD). Utilization of electron microscopy and functional phagocytic/degradative assays (such as Phrodo E. coli particle experiments) allowed detailed analysis of lysosomal structure, cargo uptake, and degradative capacity.

e) Microglia-Astrocyte Communication Analysis

Using CellChat software, the authors constructed a ligand-receptor interaction map between microglia and astrocytes, analyzed differences in communication modes between ALS subtypes (C9-ALS vs. SALS), and revealed potential molecular mechanisms affecting neuroinflammation and glial responses.

2. Main Experimental Results and Their Logical Connections

a) C9orf72 HRE Causes Decreased C9orf72 Expression in Microglia

Data showed that the hexanucleotide repeat expansion leads to C9orf72 haploinsufficiency only in microglia, while other cell types are unaffected, consistent with previous results in blood and iPSC-derived microglia.

b) Impaired Disease Response and Molecular Features in Microglia

Microglia from sporadic ALS patients transition toward reactive states, evidenced by increased expression of inflammation, lysosomal, and phagocytosis-related genes. However, microglia from C9orf72 HRE patients maintain high HM marker expression and do not show significant activation/reactive transition. Correlation with GRN-deficient cases suggests possible functional overlap. WGCNA analysis revealed that C9-ALS microglia had deficient activity in response pathways (e.g., lysosomal, apoptosis, immune response) compared to sporadic ALS.

c) Mechanistic Validation of Lysosomal-Phagocytic Pathway Impairment

Findings from xenograft models supported this: both C9-HRE type and C9orf72 knockout microglia failed to transition into reactive states (DAM/HLA), along with marked downregulation of HLA-related genes, placing familial ALS on the “deficient” rather than “activated” end of the response spectrum. In vitro experiments demonstrated that C9orf72 loss leads to enlarged and denser CTSD-positive lysosomes, while degradative efficiency of phagocytosed cargo is reduced, with accumulation in late endosomes/lysosomes—hallmarks of lysosomal dysfunction.

d) Transcriptional State Differences in Astrocytes

Astrocytes exhibited region-specific transcriptional heterogeneity, with spinal subpopulations showing more pronounced gene expression changes. SALS astrocytes upregulated reactive genes (e.g., SERPINA3), while C9-ALS astrocytes had higher levels of homeostatic and development-related genes. Damaged microglia may impair astrocyte transition and response through weakened intercellular communication.

e) Abnormal Microglia–Astrocyte Communication and Candidate Molecular Pathways

CellChat analysis identified 84 important ligand-receptor pairs, revealing ALS subtype-specific alterations in cell communication, such as upregulation of the spp1-cd44 axis in sporadic ALS (previously linked to disease progression) and gas6-mertk/axl signaling, which was specifically abnormal in C9-ALS, suggesting a potential mechanism for microglial suppression. Communication abnormalities may hinder coordinated glial responses in C9-ALS, representing new pathogenic clues.

4. Research Conclusions and Scientific Value

This study is the first to systematically reveal that C9orf72 hexanucleotide repeat expansion leads to haploinsufficiency of C9orf72 in microglia in ALS patients, suppresses their transition to disease-associated reactive states (e.g., DAM/HLA), impairs lysosomal-phagocytic function, and is accompanied by intercellular communication defects. Astrocytes also showed impaired responses, supporting the notion that ALS is a non-cell-autonomous, multi-cell-type disease. These molecular and cellular mechanism differences suggest that familial C9orf72 ALS and sporadic ALS may have essential differences in response mechanisms and therapeutic targets.

This research provides theoretical support for ALS patient stratification, pathological mechanism analysis, and precision therapy (e.g., interventions targeting lysosomal function or intercellular communication). Especially considering that familial ALS is clinically indistinguishable from sporadic ALS, uncovering such microscopic mechanism differences is of significant clinical value.

5. Research Highlights and Innovations

• Diverse Subjects and Multi-Level Systematic Methods: Combines postmortem human brain tissues, iPSC-based differentiation models, and mouse xenograft systems for comprehensive cross-validation of in vivo, in vitro, and cross-species results.
• High-Precision Single-Cell Sequencing and Integration with External Big Datasets: Clustering and subtyping allowed precise identification of microglia and astrocyte subsets, with innovative data algorithms.
• Substantive Validation of Lysosomal Dysfunction: Rare use of in vitro expansion microscopy and electron microscopy to observe microglial lysosomal ultrastructural changes, supplemented by comprehensive functional phagocytosis assays.
• First Mapping of Cellular Communication Mechanisms: Big data simulation and mapping of ALS microglia–astrocyte communication networks by CellChat, laying the foundation for subsequent drug screening and functional research.

6. Application Value and Further Inspiration

This study emphasizes the central role of microglia and astrocytes in ALS pathogenesis, providing actionable targets for stratified diagnosis and molecular-layered therapy (such as lysosomal regulation, intercellular communication modulation). It suggests that immune response activation and glial functional restoration may become directions for future precision therapy in familial ALS patients.

Moreover, this research proposed the contribution of regional heterogeneity (e.g., region-specific astrocyte gene expression in the spinal cord) to disease susceptibility, enriching the theoretical basis for regional selectivity in ALS pathology. It also sets an example for exploring common and specific mechanisms in ALS/FTD, Alzheimer’s disease, and other neurodegenerative diseases.

7. Other Important Information

The authors included extensive supplemental data supporting further validation of molecular features such as lysosomal and phagocytic signaling pathways. The study also utilizes several latest algorithms (WGCNA, MILO, CellChat), and conducted comprehensive comparisons between current mouse models, previous clinical cohorts, and in vitro cell lines, reflecting high rigor and scientific openness. Notably, the conclusion stresses the necessity of future further stratified and staged research, pointing the way for subsequent multicenter large-sample studies and drug development.

8. Summary

This top-tier research published in Nature Neuroscience for the first time elucidates in detail the significant molecular, cellular, and communication differences between familial C9orf72 ALS and sporadic ALS in microglia and astrocytes. The study not only establishes a new model for ALS pathogenesis but also provides a robust example for disease subtyping, molecular-targeted therapy, and broad immune-pathological research in neurodegenerative diseases, having immense scientific and practical value.