Single-Cell Transcriptomic and Epigenetic Profiling Highlights The Mechanisms Underlying Brain Aging

Deciphering the Epigenetic Aspects of Brain Aging at Single-cell Resolution

An improved understanding of the unresolved regulatory mechanisms underlying gene expression alterations in the multitude of cell types resident in the mammalian brain may support a deeper appreciation of aging-associated conditions (including cognitive decline, the reduced level of neural plasticity, and the increased risk of neurodegenerative disease; Mattson & Arumugam) and the development of novel preventive or curative therapeutic strategies. Multimodal single-cell epigenetic analyses may provide a means to achieve this goal, as alterations to chromatin accessibility, DNA methylation, and histone modifications represent regulators of aging (Wang et al.) and contribute to the loss of transcriptional and regulatory fidelity that impacts cell identity and function (Patrick et al.). While we possess single-cell transcriptomic atlases that depict the aging process in the mouse brain (Tabula Muris Consortium and Jin et al.), the underlying factors driving these gene expression alterations remain relatively unknown.

A previous study from Bing Ren (University of California San Diego) employing Paired-Tag technology from Epigenome Technologies which enables the simultaneous analysis of transcriptomic and epigenetic profiles in the same single cell - revealed that excitatory neurons in the mouse frontal cortex lose H3K9me3-modified heterochromatin region with increasing age, prompting increased accessibility at transposable elements and other normally repressed regions (Zhang et al.). While these findings suggested heterochromatin instability as a feature of neuronal aging, we lacked a comprehensive, multi-region, single-cell chromatin accessibility map of the aging brain to fully explore this concept.

Paired-Tag from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with comparable efficiency to single-nucleus RNA-seq/ChIP-seq assays while avoiding the need for cell sorting. Paired-Tag technology enables researchers to make significant strides in understanding gene regulation and improving disease management.

Now, researchers led by Margarita Behrens (Salk Institute) and Bing Ren have compared chromatin accessibility and DNA methylation profiles generated after analyzing single cells isolated from eight relevant brain regions (Jagust, 2013) from mice of various ages. Their bioRxiv preprint (Amaral et al.) offers profound insight into the transcriptional regulatory mechanisms underlying neuronal and glial aging in the mouse brain, linking master transcription factor loss, stress-induced reprogramming, and heterochromatin instability to age-related cell dysfunction.

Single-cell Epigenetic and Transcriptomic Analyses Provide the Fine Detail of Mouse Brain Aging

The authors of this fascinating new study applied single-cell epigenomic assays to generate chromatin accessibility and transcriptomic profiles across eight brain regions from male and female mice of 2, 9, and 18 months of age. The study employed single-nucleus assay for transposase-accessible chromatin using sequencing (ATAC-seq), 10X multiome (combining single-nucleus ATAC-seq and RNA-seq), and MERFISH spatial transcriptomics to evaluate brain regions with critical roles in cognition, memory, emotional regulation, and known vulnerabilities to age-related decline and neurodegenerative processes.

In brief, the findings of their integrative analysis highlighted i) a significant decline in three populations of progenitor cells that participated in neurogenesis and myelination; ii) widespread, concordant transcriptomic and chromatin accessibility alterations during the aging process in glial and neuronal cells; iii) the dysregulation of master transcription factors; and iv) a shift toward stress-responsive AP-1-driven transcriptional programs. Overall, these findings suggested a progressive loss of cell identity during aging. In addition, the authors identified a region- and cell-type-specific increase in accessibility at H3K9me3-associated heterochromatin domains, the activation of transposable elements, the upregulated expression of long non-coding RNAs, and increased accessibility at stress-responsive elements during the aging process; they termed these age-related epigenetic alterations as heterochromatin decay and suggested these concepts as defining features of brain aging.

In detail, the loss of the transcription factors that mediated cell identity and function represented a significant aging-related hallmark of aging in the mouse brain; for example, significant reductions in i)Lhx2Pax6SoxMef2NeuroG2

The widespread loss of heterochromatin integrity, accompanied by the dysregulation of transposable elements and non-coding RNAs, represents another critical regulator of aging. Long non-coding RNAs, particularly those located within heterochromatin-associated domains, were among the most upregulated genes. Interestingly, the authors also discovered a mechanism that may contribute to the known sex differences in brain aging and susceptibility to neurodegeneration. The study revealed that aging hotspots that overlapped with heterochromatin regions consistently occurred across autosomes in male and female mice; however, only female mice exhibited increased chromatin accessibility on the X chromosome accompanied by a loss of accessibility and expression at the pseudoautosomal region, suggesting that aging-driven chromatin reorganization extended beyond autosomal heterochromatin erosion to impact X-linked regulatory landscapes in a sex-specific manner.

Reinforcing Transcriptional Programs and Maintaining Heterochromatin to Battle Aging?

The data arising from this multiregional and multiomic single-cell study revealed how normal aging in the mouse brain occurs alongside profound changes in chromatin accessibility, transcription factor activity, transcriptional shifts, and cell identity maintenance in neuronal and glial cells; indeed, the loss of master transcription factors, stress-induced reprogramming, and heterochromatin instability appear to accompany age-related cellular dysfunction. Overall, the data suggested that the loss of transcriptional and epigenetic control over cell identity - causing cells to drift away from their defined states - represents a key aspect of the aging process, which agrees with emerging models of aging proposing a loss of epigenetic constraints that induces transcriptional drift (Patrick et al. and Yang et al.). The authors note that future efforts should focus on developing targeted strategies to reinforce lost transcriptional programs and maintain heterochromatin, thereby preserving cognitive function and delaying the progression of neurodegeneration.

Paired-Tag from Epigenome Technologies generates joint epigenetic and gene expression profiles at the single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Furthermore, Epigenome Technologies offers a range of single-cell products and services tailored to meet the majority of research requirements. As such, applying Paired-Tag technology may enable giant leaps forward in understanding gene regulation and complement the findings of this exciting study.

For more on how single-cell transcriptomic and epigenetic profiling can highlight potentially targetable mechanisms underlying brain aging, see bioRxiv, May 2025.