Astrocytes shed their supportive role and unlock hidden neurogenic potential, offering new hope for brain injury recovery through cutting-edge epigenetic reprogramming.
Study: DNA methylation controls stemness of astrocytes in health and ischaemia. Image Credit: Christoph Bock, Max Planck Institute for Informatics
In a recent study published in the journal Nature, researchers in Germany investigated the chromatin accessibility, transcriptome, and methylome of neural stem cells, their progeny, and astrocytes in the healthy and ischemic (with impaired blood flow) adult mouse brain. They found that ischemic injury induces astrocytes to modify their methylome to a stem cell state, involving the methylation of astrocyte-specific genes and the demethylation of stem cell-associated genes, a process dependent on DNA methyltransferase 3 alpha (DNMT3A).
Background
Adult neurogenesis once thought to be impossible in mammals, is now known to occur in specific brain regions like the dentate gyrus and the ventricular–subventricular zone (vSVZ). In the vSVZ of mice, specialized astrocytes function as neural stem cells (NSCs). NSCs differentiate into transit-amplifying progenitors (TAPs) that produce neuroblasts, which migrate to the olfactory bulb to become neurons while also giving rise to various glial cells. NSCs alternate between quiescent and active states, with gene expression profiles showing that quiescent NSCs closely resemble non-neurogenic astrocytes found in the striatum and cortex. This similarity raises the possibility that these astrocytes could be reprogrammed to generate neurons, a process that involves dynamic changes in DNA methylation, a promising avenue for regenerative medicine.
Single-cell ribonucleic acid sequencing allows the detailed tracking of changes in gene expression along the neurogenic lineage. Studies suggest that astrocytes in the striatum may acquire neurogenic potential after injury, offering hope that latent neurogenic abilities could be unlocked in other astrocytes, contributing to neural repair and regeneration after damage. In the present study, researchers used single-cell nucleosome, methylome, and transcriptome sequencing (scNMT-seq) to investigate whether gene expression changes in NSCs are driven by epigenetic alterations, specifically focusing on the interplay between DNA methylation and gene regulation.
About the study
In the present study, male mice of various lines, including C57BL/6N and TiCY, aged two to four months, underwent tamoxifen injection to induce Cre recombination in NSCs before a bilateral common carotid artery occlusion. Single-cell suspension and fluorescence-activated cell sorting were performed on isolated brain regions, with specific antibodies used to label and sort different cell populations. A miniaturized scNMT-seq protocol was employed to profile the transcriptome and epigenome of single cells. Dnmt3a was knocked out in striatal cells, and immunohistochemistry was conducted to quantify cells in ischemic conditions. The study also included a neurosphere assay to evaluate neurogenic potential. Transcriptomic and epigenomic data were analyzed using Seurat and MethSCAn tools. Statistical methods were applied to assess differential methylation and gene expression, along with transcription factor motif enrichment and gene ontology term identification.
Results and discussion
Integration of single-cell transcriptomes revealed a continuum of cell states, including dormant, quiescent, and active NSCs, TAPs, neuroblasts, neurons, and oligodendrocytes. Dynamic changes in deoxyribonucleic acid (DNA) methylation and chromatin accessibility were observed along the lineage. Quantification of DNA methylation and chromatin accessibility profiles indicated the robustness of the epigenomic data.
Variably methylated regions (VMRs) were found to predict gene expression better than promoter regions. In NSC lineage progression, genes with increased methylation downstream of the transcription start site (TSS) showed reduced expression. This indicates that DNA methylation in this region may silence gene expression, while promoter methylation is less dynamic than that of enhancers.
Distinct methylation patterns were observed between dormant (qNSC1) and active NSCs (qNSC2). qNSC1 cells, resembling striatal astrocytes, displayed a methylome that transitions to an NSC methylome in qNSC2. Low-methylation regions (LMRs) in astrocytes were linked to genes involved in amino acid transport, indicating their role in astrocyte identity. In contrast, LMRs in qNSC2 cells were enriched for genes regulating differentiation and stem cell functions, highlighting a significant epigenetic divide despite similar transcriptomes. Gene accessibility showed a gradual increase during NSC activation, suggesting that DNA methylation is key in distinguishing stem cell functions from common astrocytes.
Ischemia triggered vSVZ and striatal astrocytes to adopt an NSC methylome characterized by high methylation of astrocyte LMRs and low methylation of NSC LMRs, indicating increased neurogenesis. After 21 days post-ischemia, astrocytes reverted to a naive methylation profile. However, the study also identified a small subset of astrocytes that retained an NSC methylome or showed incomplete reversion, suggesting a more complex dynamic of methylation changes post-injury. A new cluster of reactive astrocytes emerged, with varied gene expression linked to neurogenic and reactive phenotypes. Interferons were identified as crucial for establishing specific DMRs post-ischemia, indicating their key role in guiding the epigenetic changes necessary for astrocyte reprogramming.
Control mice showed a significant increase in neuroblasts following ischemia, while Dnmt3a-deficient mice showed a marked reduction in this response, with neuroblast generation being nearly absent. These findings suggest that methylome remodeling mediated by DNMT3A is essential for activating the neurogenic potential in striatal astrocytes.
Conclusion
In conclusion, the study reveals that DNA methylation plays a crucial role in enabling astrocytes to acquire stem cell functions in the adult mouse brain. Usually, astrocyte methylomes lock them in their supporting role, but ischemic injury triggers methylation changes that allow neurogenesis. This dynamic nature of DNA methylation may serve as a blueprint for future cell states, guiding the differentiation process. These findings suggest that DNA methylation is dynamic and may guide future cell states, offering potential therapeutic avenues for nervous system repair and cancer treatment. In the future, exploring methylation manipulation could potentially enhance cell reprogramming efficiency.
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