Disruption of Nucleolar Integrity and Dysregulation of RNA Splicing in C9orf72-FTD/ALS

Background and Research Motivation

The hexanucleotide repeat expansion (GGGGCC)n in the C9orf72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Studies have shown that these repeat sequences not only form toxic RNA aggregates but also produce neurotoxic dipeptide repeat (DPR) protein aggregates through atypical translation, particularly poly-glycine-arginine (Poly-GR). RNA processing abnormalities induced by these pathological features, such as RNA mis-splicing, are widespread issues in ALS and FTD patients. Although previous research has revealed some mechanisms of interaction between RNA-binding proteins (RBPs) and these repeat RNAs, it is not yet clear how these interactions lead to global splicing dysregulation.

In this study, the researchers attempt to uncover the effects of (GGGGCC)n repeat RNA on nucleolar phase separation properties and dynamics, further exploring how these changes cause global RNA splicing defects and induce neurotoxicity. This work not only enriches our understanding of the pathogenesis of C9-FTD/ALS but also may provide new directions for future biomarkers or therapeutic targets.

Research Origin and Publication

This paper, authored by Rong Wu and several other scientists from Johns Hopkins University and other internationally renowned research institutions, was published on October 23, 2024, in the internationally renowned neuroscience journal Neuron. This study provides a new perspective on the potential pathogenic mechanisms of ALS and FTD, particularly in the deep exploration of nucleolar and RNA splicing dysregulation.

Research Process and Methods

1. Research Design and Experimental Materials
   The researchers first utilized plasmid transfection and gene expression induction techniques to establish HEK293T cell lines and iPSC-derived neuronal models containing (GGGGCC)n repeat sequences. Through RNA affinity purification and mass spectrometry analysis, they detected the types of proteins near the repeat sequences and their interactions, further confirming the colocalization of nucleolar-specific proteins (such as SRRM2) with the repeat sequences.

2. Nucleolar Phase Separation Properties and Dynamics
   Through immunofluorescence colocalization detection, the study found that (GGGGCC)n repeat sequence RNA colocalizes with the nucleolar protein SRRM2, significantly altering nucleolar size and dynamic properties. Especially in the cell models, the accumulation of repeat RNA leads to changes in nucleolar phase separation characteristics, resulting in gel-sol transitions that inhibit its normal dynamics.

3. Multi-Step Data Analysis and Validation of Global RNA Splicing Defects
   At the molecular mechanism level, researchers used RNA sequencing and data analysis to discover that neurons containing repeat sequences exhibit widespread RNA splicing abnormalities, such as exon skipping and intron retention. Notably, knockdown experiments revealed that SRRM2 or SON deficiency triggers widespread splicing events, significantly increasing exon skipping and intron retention. These splicing defects were also validated in C9-FTD/ALS patient tissues.

4. Confirmation of the Relationship between Nucleolar Damage and Neurotoxicity
   To further investigate the relationship between nucleolar damage and neurotoxicity, researchers used lactate dehydrogenase (LDH) release assays to detect neuron death. Results indicated that the loss of SRRM2 or SON leads to neuronal cell death, suggesting that loss of nucleolar function causes neurotoxicity. Furthermore, the researchers successfully reduced neurotoxicity induced by (GGGGCC)n overexpression by restoring SRRM2 expression through a drug induction system, further validating the importance of nucleolar function.

Main Research Results

1. Interaction between Repeat RNA and Nucleolar Proteins
   (GGGGCC)n repeat sequence RNA colocalizes with the nucleolar protein SRRM2, leading to dysregulation of SRRM2 liquid-liquid phase separation, thereby affecting nucleolar formation and dynamic characteristics.

2. Global RNA Splicing Abnormalities Induced by Repeat Sequences
   Nucleolar damage induced by repeat sequences causes global RNA splicing defects, mainly manifested as exon skipping and intron retention. Analysis shows that splicing defects due to SRRM2 and SON deficiency concentrate in GC-rich regions, suggesting these regions may be specific binding targets for nucleolar proteins.

3. Effect of Poly-GR on Nucleolar Protein Aggregation
   In mouse models, Poly-GR co-aggregates with SRRM2, further exacerbating nucleolar dysfunction. Similar phenomena were observed in patient neurons, indicating that Poly-GR may play an important role in C9-FTD/ALS pathology.

4. Relief of Neurotoxicity through Functional Restoration of Nucleolar Proteins
   The study demonstrated that inducing the restoration of SRRM2 expression significantly reduces the neurotoxicity caused by (GGGGCC)n overexpression. This finding offers a new approach for alleviating ALS and FTD pathology through restoring nucleolar integrity in the future.

Conclusion and Research Value

This research, through systematic molecular and cellular experiments, reveals the critical role of nucleolar dysfunction in C9-FTD/ALS, particularly in impacting RNA splicing dysregulation. The study indicates that nucleolar dysfunction caused by the co-aggregates of (GGGGCC)n repeat sequences and Poly-GR may represent a convergent toxic pathway in this disease. Global splicing inhibition caused by nucleolar damage may be a common mechanism in various neurodegenerative diseases. Especially, the loss of SRRM2 and the associated splicing dysregulation in GC-rich regions emphasize the importance of specific sequences and regions in maintaining nucleolar function.

From an application perspective, the research provides a potential new direction for treating C9-FTD/ALS by targeting the nucleolus or restoring SRRM2 function. Meanwhile, these findings broaden our understanding of RNA processing abnormalities in neurodegenerative diseases, especially the molecular mechanisms of splicing defects, which may aid in developing therapeutic strategies for other similar diseases.