Characteristics of Premature Cellular Senescence in Alzheimer’s Disease: Application of Single-Nucleus Transcriptomics

Research Background and Objectives

Alzheimer’s disease (AD) is the most common type of dementia in the elderly, characterized by extracellular deposition of β-amyloid protein and intracellular neurofibrillary tangles. Other pathological features include an increase in microglia, astrocytes, mitochondrial and lysosomal dysfunction, and neurodegeneration. Aging is the greatest risk factor for AD and is closely related to cellular senescence. Cellular senescence refers to the irreversible cell cycle arrest state after cells reach the so-called “Hayflick limit.” However, under chronic stress (such as oxidative stress and mitochondrial dysfunction), both proliferative and non-proliferative cells can exhibit premature cellular senescence. Cellular senescence is often used to describe the cell state transformation through the secretion of various factors, known as the senescence-associated secretory phenotype (SASP).

Although there is previous evidence of premature senescence in glial cells in AD patients, their overall burden, the most affected cell types, and the mechanisms triggering senescence have not been fully described. Therefore, this study, in collaboration with Imperial College London and several international research institutions, aims to comprehensively describe the characteristics of premature cellular senescence in Alzheimer’s disease, explore its mechanisms, and provide a basis for future treatment plans.

Research Source and Authors

This study was jointly completed by 18 scholars, including Nurun N. Fancy, Amy M. Smith, Alessia Caramello, from the Brain Sciences Department of Imperial College London, the Centre for Brain Research of the University of Auckland, the Department of Psychiatry of the University of Geneva, and other research institutions. The paper was published in the journal Acta Neuropathologica in 2024, with the submission date of January 4, 2024, the revision date of March 11, 2024, and the acceptance date of March 28, 2024.

Research Methods and Processes

Overview of Research Process

This study used Imaging Mass Cytometry (IMC) and Single-Nucleus RNA Sequencing (snRNA-seq) technology to analyze over 200,000 nuclei and obtain data from the midbrain and cortical tissues of non-disease control (NDC) and AD donors. The research process included: sample processing, IMC and snRNA-seq data collection, data analysis, and validation.

Sample Processing

The research samples came from two groups of donors, one group included 10 non-disease controls (NDC) (Braak stages 0-II), and the other group included 10 AD patients (Braak stages V-VI). These samples were labeled using Imaging Mass Cytometry, particularly with immunostaining markers for β-galactosidase (GLB1) and DNA damage repair protein p16INK4a (p16).

Data Collection and Analysis

Using Imaging Mass Cytometry technology, the study found that compared to NDC, the GLB1-positive microglia, oligodendrocytes, and astrocytes in AD increased by 4.1, 4.6, and 4 times respectively, and p16-positive microglia increased by 1.6 times. Subsequently, using single-nucleus RNA sequencing technology, detailed gene expression analysis was performed on seven major brain cell types in midbrain and cortical tissues.

Gene Set Enrichment Analysis (GSEA) and Pseudotime Trajectory Analysis were used to describe premature senescence characteristics related to DNA double-strand breaks (DSBs), mitochondrial dysfunction, and endoplasmic reticulum (ER) stress, and validate the phenomena observed through independent AD snRNA-seq datasets.

Main Results

Increased Expression of Cellular Senescence Markers

Imaging Mass Cytometry results showed that in AD, compared to NDC, the expression of GLB1 and p16 markers significantly increased in microglia, oligodendrocytes, and astrocytes. It was also observed that the phagocytic pathway of microglia in AD was downregulated, indicating a reduced ability to clear β-amyloid.

Association of Premature Senescence with β-Amyloid

To further understand the relationship between β-amyloid burden and cellular senescence, the study found that in the brain tissue of AD donors, more than 25% of microglia near β-amyloid plaques simultaneously expressed GLB1 and p16, whereas only 3% of microglia far from plaques showed these markers.

Results of Single-Nucleus RNA Sequencing Analysis

Single-nucleus RNA sequencing data revealed that microglia in AD exhibited significant premature senescence-related gene expression, such as ATM, RB1, NFATC2, and GLB1. Particularly, microglia in AD showed upregulation in pathways related to protein synthesis, metabolic regulation, and endoplasmic reticulum stress. These data suggest that premature senescence of microglia in AD may be a prominent phenomenon.

Conclusion and Significance

The study results indicate that in Alzheimer’s disease, there is a high burden of premature senescent microglia, closely related to disease progression. Microglia in AD not only exhibit significant markers of premature senescence but also show gene expression characteristics associated with β-amyloid burden. This provides a new perspective for future research, suggesting that microglia may be the primary therapeutic target in AD, and that removing senescent cells may help reduce pathological burden and cognitive impairment.

These findings also support that premature senescence may play a key role in the progression of AD, providing a scientific basis for designing therapeutic strategies based on the removal of senescent cells.

Research Highlights

1. Important Findings: The study revealed a significant increase in the burden of premature cellular senescence in the brains of AD patients, particularly in microglia.
2. Senescence Markers: Detailed analysis of the expression differences of cellular senescence markers through Imaging Mass Cytometry and single-nucleus RNA sequencing.
3. Mechanism Construction: The study thoroughly described premature senescence mechanisms related to DNA double-strand breaks, mitochondrial dysfunction, and endoplasmic reticulum stress.
4. Potential Therapeutic Targets: The research data support microglia as the primary therapeutic target for treating Alzheimer’s disease, providing important evidence for designing future treatment plans.

Additional Valuable Information

This study not only validated the results of previous independent datasets but also pointed out further research directions, such as more specifically defining the gene expressions driving pathology. Particularly, it focused on the impact of β-amyloid-related stress on microglia and how to design therapeutic strategies affecting their senescence pathways.