Can Single-Cell Epigenetic Analyses Help to Understand Cognitive Decline and Disease Susceptibility?

Aging represents the greatest risk factor for the development of neurodegenerative diseases (Hou et al.); as such, the aging of the human brain has been linked to physiological and cognitive decline and the associated diminished quality of life (Golomb et al. and Jack et al.). Research on vulnerable brain regions has revealed loss of function and atrophy in the hippocampus (Golomb et al., Jack et al., and Bettio et al.), which plays important roles in learning and memory. While studies have defined age- and dysfunction-related alterations to gene expression profiles in the human brain in general and the hippocampus specifically, exploring epigenetic regulation and higher-order genome organization at the single-cell level may provide a more complete view and deeper understanding of cognitive decline and disease susceptibility.

Researchers from the laboratories of Xiangmin Xu and Bing Ren recently undertook a single-cell multiomic study of the aging human adult hippocampus, applying a wide range of assays to explore chromatin accessibility, DNA methylation, and 3D genome organization. Overall, this exciting new Science paper reveals that the human hippocampus undergoes age-related alterations in cell composition and regulatory mechanisms and also provides a framework for understanding age-related memory decline and increased vulnerability to neurodegenerative disease and for designing novel therapeutic approaches (Zemke, Lee, and Mamde et al.).

Paired-Tag technology from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Could an in-depth analysis of the histone modification and transcriptomic profiles of the same single cells in the aging hippocampus afforded by the integration of Paired-Tag have provided more profound insight into how the alterations in epigenetic mechanisms over time impact the gene expression profiles that control cell identity? Could this information also add another layer of insight to guide future studies aimed at promoting healthy brain aging and developing novel neurodegenerative disease treatment strategies?

Single-Cell Epigenetic Analyses Depict Age-related Alterations to Crucial Brain Cells and Chromatin Conformation

Briefly, the authors discovered linear and non-linear dynamic alterations in gene regulation across hippocampal cell types during normal aging, with a major transition around midlife (between ages 50 and 75) that correlated with age-associated epigenomic remodeling. As such, the study highlighted significant remodeling of the immune cell landscape in the brain, which manifested as the age-dependent replacement of embryonic yolk sac-derived brain-resident microglia with a population of microglia whose epigenetic profile greatly resembled peripheral blood monocyte-derived microglia. Importantly, gene expression or chromatin accessibility analysis could not distinguish these two cell types alone and, as such, failed to identify this aging-associated cell replacement phenomenon; however, DNA methylation patterns (which preserve a memory of cell lineage; Hon et al.) represented the defining factor that helped to highlight the presence of these distinct microglial lineages.

[! Multi-panel image showing single-cell embeddings of microglial and immune populations, and tracks of epigenetic states](img/image0.png Panels D–F integrate single-nucleus DNA methylation and browser data to show that in aged brains starting around age 50, peripheral monocytes infiltrate the central nervous system and trans-differentiate into microglia-like states. Specifically, Panel D displays the mCG fraction of differentially methylated regions (DMRs) between Micro1 and Micro2 cells alongside peripheral blood monocytes; Panel E provides WashU Epigenome Browser screenshots of the HOXA cluster illustrating matching mCG fractions and chromatin accessibility between Micro2 and monocytes; and Panel F displays a UMAP projection of 5 kb mCG profiles comparing hippocampal cells, peripheral blood, skin, muscle fibroblasts, and infant microglia to track these lineage relationships. From (Zemke, Lee, and Mamde et al.)

The authors note that this level of epigenetic analysis may now support future investigations into their relative associations with neurodegenerative disease processes within the human brain. Importantly, the replacement peripheral blood monocyte-derived microglia exhibited epigenetic, transcriptional, and 3D chromatin conformation features suggesting a pro-inflammatory profile; therefore, this aging-associated cell replacement phenomenon may drive the chronic neuroinflammation thought to underlie many diseases/disorders. Importantly, while previous research supports these exciting findings (Kim et al. and Bouzid et al.), the mechanistic triggers that signal for these immune cells to enter the brain and proliferate during aging remain unexplored. Taken together with emerging evidence from other studies, these findings challenge the model that yolk sac-derived microglia maintain the microglial population across a human lifespan (Ginhoux et al.).

Remaining at the cell level, the authors also discovered a significant decline in the number of multifunctional hippocampal astrocytes during aging, including those that support synaptic signaling and maintain the blood-brain barrier. The loss of hippocampal astrocytes occurred alongside the acquisition of a gene expression profile that suggested reduced mitochondrial energy production and increased cellular stress, suggesting metabolic dysfunction as the mechanism behind cell loss. Notably, these findings disagree with previous cross-species research that reported a lack of substantial astrocyte loss during aging (Labarta-Bajo & Allen and Verkhratsky et al.) and thereby provide the impetus for studies into the triggers of hippocampal astrocyte attrition during normal/pathological aging.

[! Multi-panel image showing single-cell embeddings of astrocytes and pathways associated with age-associated astrocyte cCRE](img/image1.png Panels A through G reveal that aging drives a significant loss of critical support cells like astrocytes, oligodendrocyte progenitor cells, and endothelial cells in the human hippocampus, compromising blood-brain barrier integrity and synaptic regulation. Scatter plots and confocal imaging of hippocampal CA3 tissue using the astrocytic marker ALDH1L1 demonstrate a substantial reduction of astrocytes in aged donors compared to young donors. Complementary Gene Ontology analyses show the functional categories of age-correlated genes that decrease or increase over time in astrocytes. Finally, an expression heatmap correlates these transcriptional shifts with corresponding mCG DNA methylation levels across gene bodies, illustrating the coordinated epigenetic remodeling that accompanies cellular decline during brain aging. From (Zemke, Lee, and Mamde et al.)

Finally, the authors highlighted significant alterations at the chromatin conformation level: they observed the global "erosion" of 3D genome organization in multiple hippocampal cell types during aging. These changes included reduced integrity of topologically associating domains and increased trans-chromosomal interactions, which coincided with other epigenetic alterations and the observed shifts in gene expression profiles.

Providing the Basis of Future Research into Aging and Therapeutic Development?

Overall, this fascinating multi-omic single-cell epigenetic study of the aging hippocampus highlights significant alterations in cell composition – exemplified by immune cell exchange and astrocyte loss - and chromatin conformation that may underpin the loss of memory and increased neurodegenerative disease vulnerability known to occur during the normal aging process. These data also provide insight into how altered gene regulatory programs drive cell-type-specific aging phenotypes in the human brain – including age-related neuroinflammation and synaptic dysfunction – and provide a framework for the future dissection of aging phenotypes at the single-cell level, which may provide us with the ability to promote healthy brain aging and target age-associated neurodegenerative disease.

The implementation of Paired-Tag technology from Epigenome Technologies, which generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays, could provide deeper insight into these research aims. Could the simultaneous single-cell analysis of histone modifications and transcriptomic profiles have contributed to the wide range of innovative techniques employed and added further depth to this fascinating study of hippocampal aging?