Correlation between PTDP-43 Levels and Cell-Specific Molecular Alterations in the Prefrontal Cortex of C9orf72 ALS/FTD Patients

Background Introduction

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two types of progressive neurodegenerative diseases. Their common feature is the loss of specific neuronal populations in the central nervous system (CNS). Although these two diseases differ symptomatically, they share common genetic and neuropathological features, particularly the G4C2 hexanucleotide repeat expansion mutation in the C9orf72 gene, which is considered the most common genetic cause of both ALS and FTD.

ALS primarily manifests as the degeneration of motor neurons, leading to muscle weakness and respiratory failure, while FTD involves the degeneration of the frontal and temporal cortex, resulting in a gradual decline in cognitive function. In both diseases, the phosphorylation, mislocalization, and aggregation of TAR DNA-binding protein 43 (TDP-43) are hallmark neuropathological features. TDP-43 is a widely expressed nuclear protein involved in RNA metabolism, including RNA splicing and mRNA stability. Studies have shown that the pathological burden of TDP-43 correlates with the degree of degeneration of affected cell populations in ALS and FTD patients.

However, studying changes in the brains of patients with neurodegenerative diseases during disease progression is challenging due to the difficulty in obtaining live brain tissue samples. Therefore, this study aims to reveal molecular changes in different cell types during disease progression by analyzing single-cell multi-omics data from the prefrontal cortex of C9orf72 ALS/FTD patients, especially cell-specific changes related to PTDP-43 levels.

Source of the Paper

This paper was a collaborative effort by research teams from multiple institutions, including Emory University School of Medicine, Mayo Clinic, and the University of Florida. The main authors include Hsiao-Lin V. Wang, Jian-Feng Xiang, Victor G. Corces, and Zachary T. McEachin. The paper was published in the journal PNAS (Proceedings of the National Academy of Sciences) on February 25, 2025, titled “PTDP-43 levels correlate with cell type–specific molecular alterations in the prefrontal cortex of C9orf72 ALS/FTD patients.”

Research Workflow

1. Sample Acquisition and Grouping

This study used postmortem brain tissue samples from C9orf72 ALS/FTD patients and healthy controls, primarily analyzing cells from the prefrontal cortex (Brodmann area 9). The samples were divided into two groups: 19 C9orf72 ALS/FTD patients and 7 age/gender-matched healthy controls. All C9orf72 ALS/FTD patients had clinical diagnoses of ALS and/or FTD and exhibited varying degrees of cognitive impairment.

To investigate the impact of PTDP-43 levels on disease progression, researchers categorized C9orf72 ALS/FTD patients into three groups based on PTDP-43 levels: PTDPneg (PTDP-43 levels below 100), PTDPmed (PTDP-43 levels between 100 and 2000), and PTDPhigh (PTDP-43 levels above 2000).

2. Single-Cell Multi-Omics Analysis

Researchers used 10x Genomics Chromium single-cell multi-omics technology to simultaneously analyze the transcriptome (snRNA-seq) and epigenome (snATAC-seq) of single nuclei from the prefrontal cortex. By analyzing samples from the Emory and Mayo brain banks, researchers obtained multi-omics data from 34,874 and 53,331 single nuclei, respectively.

Data analysis utilized the ArchR and Seurat software packages for quality control, dimensionality reduction, and batch effect correction. Through Uniform Manifold Approximation and Projection (UMAP) and unsupervised clustering, researchers identified 31 cell clusters in the Emory samples and 20 cell clusters in the Mayo samples. These cell clusters were further subdivided into major cortical cell types, including excitatory neurons, inhibitory neurons, astrocytes, microglia, oligodendrocytes, oligodendrocyte precursor cells, and endothelial cells.

3. Differential Gene Expression and Chromatin Accessibility Analysis

Researchers conducted differential gene expression (DEGs) and chromatin accessibility (Differentially Accessible Regions, DARs) analyses for each cell type. The results showed that a large number of DARs appeared in non-neuronal cells (especially oligodendrocytes) in the PTDPhigh group, indicating that changes in transcription factor binding sites might be a hallmark of cellular dysfunction in the late stages of the disease.

Additionally, researchers found a large number of differentially expressed genes in non-neuronal cells in the PTDPhigh group. These genes are mainly involved in synaptic transmission, myelination, and microtubule protein coding. By analyzing the association between chromatin accessibility and gene expression, researchers identified some key genes related to disease progression, such as CCK, SUN2, and SOX10.

4. Oligodendrocyte Maturation Impairment

The study found that a large number of immature premyelinating oligodendrocytes appeared in oligodendrocytes in the PTDPhigh group. These cells are typically transiently present in the adult brain. This suggests that the maturation process of oligodendrocytes is hindered in the late stages of the disease, possibly due to cytoplasmic accumulation of PTDP-43 or loss of nuclear TDP-43.

5. Changes in Microglia and Astrocytes

The study found that microglia exhibit a loss of neuronal surveillance function early in the disease, which further intensifies in the late stages. Additionally, astrocytes show upregulation of NEAT1 and RPS6KA2 genes in the late stages of the disease. These genes are associated with RNA retention and nuclear protein phosphorylation and may be involved in the pathological processes of the late stages of the disease.

Research Conclusions

Through single-cell multi-omics analysis, this study reveals molecular changes in different cell types in the prefrontal cortex of C9orf72 ALS/FTD patients during disease progression. The study finds that the accumulation of PTDP-43 is associated with extensive changes in chromatin accessibility and gene expression in non-neuronal cells (especially oligodendrocytes). Moreover, the maturation impairment of oligodendrocytes, the loss of neuronal surveillance function in microglia, and the abnormal activation of astrocytes are important features of disease progression.

These findings not only deepen our understanding of the pathogenic mechanisms of C9orf72 ALS/FTD but also provide potential targets and timing for future therapeutic interventions.

Research Highlights

1. Single-Cell Multi-Omics Analysis: For the first time, this study combines single-cell transcriptome and epigenome analyses to comprehensively reveal molecular changes in different cell types in the prefrontal cortex of C9orf72 ALS/FTD patients.
2. Classification Study Based on PTDP-43 Levels: By grouping patients according to PTDP-43 levels, researchers were able to systematically study cellular and molecular changes at different stages of the disease.
3. Oligodendrocyte Maturation Impairment: The study found that the accumulation of PTDP-43 leads to oligodendrocyte maturation impairment, which may be an important cause of neuronal degeneration in the late stages of the disease.
4. Abnormal Activation of Microglia and Astrocytes: The study reveals different responses of microglia and astrocytes in the early and late stages of the disease, providing new insights into the role of neuroinflammation in neurodegenerative diseases.

Significance of the Study

This study not only provides new insights into the pathogenic mechanisms of C9orf72 ALS/FTD but also offers potential targets for future treatment strategies. By revealing molecular changes in different cell types during disease progression, researchers have laid the groundwork for developing treatments targeting specific cell types. Additionally, this study demonstrates the powerful potential of single-cell multi-omics technology in neurodegenerative disease research, providing new directions for future studies.