Tegowski M, Prater AK, Holley CL, Meyer KD. Single-cell m6A profiling in the mouse brain uncovers cell type-specific RNA methylomes and age-dependent differential methylation. Nat Neurosci. 2024 Dec;27(12):2512-2520. Epub 2024 Sep 24 PubMed.
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The University of Queensland
In recent years, scientists have started to be investigate the role of m6A-RNA regulation in brain aging and Alzheimer’s disease. Where, and how, m6A changes in the transcriptome are critical questions that have primarily been approached using bulk tissue analyses, coupled with an antibody-based immunoprecipitation, in animal models and human postmortem brain (Shafik et al., 2021; Huang et al., 2023; Castro-Hernández et al., 2023). This paper by Tegowski et al. reports the first instances of single-nucleotide and single-cell m6A-transcriptomic mapping of the mouse brain. The authors pioneered a tool for detecting m6A in cellular RNA that uses a modified RNA-editing enzyme, APOBEC, fused to the m6A-binding YTH domain. They call the method “DART,” ordeamination adjacent to RNA modification targets. Now, by generating a DART transgenic mouse line, the authors could perform in vivo labelling of m6A followed by single-cell sequencing (scDART-Seq) analysis, a much-anticipated tool for revealing the distribution of m6A in highly heterogenous tissue such as the brain.
Indeed, the scDART-Seq analysis revealed cell-specific regulation of m6A in the brain, particularly clustered within glutamatergic neuronal populations of different cortical layers. Changes in methylation in the aged mouse cortex also most prominently affected glutamatergic neurons, including transcripts associated with neurodegenerative disease. While neuronal cells generally had high levels of methylated sites, amongst all cell types in the mouse cortex, microglia appeared to be particularly deprived of m6A. As these are the brain-resident immune cells, this novel finding triggers questions regarding m6A and reactive microglia in neurodegenerative disease, where these cells undergo morphological, molecular, and functional remodelling in response to brain challenges.
Single-cell m6A-profiling datasets are valuable resources for the neuroscience community. The next exciting adventure for the DART mice would be to test distinct behavioural exposures or stressors and determine how the brain RNA methylome changes in cell-type- and stimulus-dependent manner in the context of health and disease.
References:
Shafik AM, Zhang F, Guo Z, Dai Q, Pajdzik K, Li Y, Kang Y, Yao B, Wu H, He C, Allen EG, Duan R, Jin P. N6-methyladenosine dynamics in neurodevelopment and aging, and its potential role in Alzheimer's disease. Genome Biol. 2021 Jan 5;22(1):17. PubMed.
Huang H, Song R, Wong JJ, Anggono V, Widagdo J. The N6-methyladenosine RNA landscape in the aged mouse hippocampus. Aging Cell. 2023 Jan;22(1):e13755. Epub 2022 Dec 9 PubMed.
Castro-Hernández R, Berulava T, Metelova M, Epple R, Peña Centeno T, Richter J, Kaurani L, Pradhan R, Sakib MS, Burkhardt S, Ninov M, Bohnsack KE, Bohnsack MT, Delalle I, Fischer A. Conserved reduction of m6A RNA modifications during aging and neurodegeneration is linked to changes in synaptic transcripts. Proc Natl Acad Sci U S A. 2023 Feb 28;120(9):e2204933120. Epub 2023 Feb 22 PubMed.
View all comments by Jocelyn WidagdoVIB-Center for Molecular Neurology
This study is very exciting and opens new ways to assess cell heterogeneity in the brain that were not possible before. We have, and are, learning a lot from single-cell transcriptomes, and these new technologies would allow us to dive more deeply into the mechanism governing cellular diversity in the healthy and diseased brain.
It is striking to see how different neuronal subtypes have differentially methylated RNAs, and it would be very interesting to link that to protein expression to see what would be the functional impact.
It is also interesting to see a generally lower level of methylation in microglia. This is, of course, only exploring cells in their steady state, and given the reactive nature of microglia, I would be very intrigued to see how this would change in disease conditions, as well as what role RNA methylation may play in different cell states.
The observation regarding the differential methylation of APP RNA in aging is very thought-provoking. This could indicate that over time, the dosage of APP may change, regardless of the levels of RNA expression, which could subsequently be contributing to amyloid accumulation in late-onset AD patients.
I feel that both the findings on microglia and APP deserve follow-up studies because they bring new insights into the biology of AD.
View all comments by Renzo MancusoUniversity of Alabama at Birmingham
Over the past decade, there have been significant technological advancements in single-cell transcriptome and epigenome profiling, as well as in spatial transcriptomics and proteomics. These methods have been crucial in understanding altered gene regulation mechanisms in Alzheimer’s disease (Anderson et al., 2023; Morabito et al., 2021; Mathys et al., 2024). Recently, global profiling of the epitranscriptome at the single-cell level has become possible with techniques like scDART-Seq, developed by the Meyer lab (Tegowski et al., 2022). In this new study, scDART-Seq was applied in vivo using a transgenic mouse model, enabling precise and sensitive profiling of m6A at the single cell level across any tissue and at any time.
In applying this method to mouse brain tissue, the authors found that m6A levels were generally consistent across most cell types, except for microglia, which exhibited significantly lower levels. As stated by the authors, this finding aligns with previous research indicating altered m6A levels in activated microglia populations, and it would be valuable to replicate these analyses in an Alzheimer’s disease mouse model (Li et al., 2021). Furthermore, the study showed that m6A modifications could distinguish subclusters within excitatory neurons independently of gene expression changes, particularly affecting genes involved in synaptic transmission. As noted by the authors, this could be attributed to the role of m6A in mRNA localization to neurites, essential for proper synapse function. It would be interesting to integrate these findings with spatial measurements of these mRNAs and their protein products to determine if this is indeed disrupting localization or local mRNA translation at the synapse.
Additionally, the study found age-related hypermethylation in glutamatergic neurons, while App m6A levels decreased with age specifically in this cell type. Understanding how these changes impact APP function, localization, or translation efficiency, particularly in the context of neurodegeneration, will be an important area for future research.
These technological advances in single-cell profiling enhance our ability to interpret how changes in the transcriptome, epitranscriptome, and epigenome in specific cell types contribute to neurodegeneration.
References:
Anderson AG, Rogers BB, Loupe JM, Rodriguez-Nunez I, Roberts SC, White LM, Brazell JN, Bunney WE, Bunney BG, Watson SJ, Cochran JN, Myers RM, Rizzardi LF. Single nucleus multiomics identifies ZEB1 and MAFB as candidate regulators of Alzheimer's disease-specific cis-regulatory elements. Cell Genom. 2023 Mar 8;3(3):100263. Epub 2023 Feb 2 PubMed.
Li Q, Wen S, Ye W, Zhao S, Liu X. The potential roles of m6A modification in regulating the inflammatory response in microglia. J Neuroinflammation. 2021 Jul 5;18(1):149. PubMed.
Mathys H, Boix CA, Akay LA, Xia Z, Davila-Velderrain J, Ng AP, Jiang X, Abdelhady G, Galani K, Mantero J, Band N, James BT, Babu S, Galiana-Melendez F, Louderback K, Prokopenko D, Tanzi RE, Bennett DA, Tsai LH, Kellis M. Single-cell multiregion dissection of Alzheimer's disease. Nature. 2024 Aug;632(8026):858-868. Epub 2024 Jul 24 PubMed.
Morabito S, Miyoshi E, Michael N, Shahin S, Martini AC, Head E, Silva J, Leavy K, Perez-Rosendahl M, Swarup V. Single-nucleus chromatin accessibility and transcriptomic characterization of Alzheimer's disease. Nat Genet. 2021 Aug;53(8):1143-1155. Epub 2021 Jul 8 PubMed.
Tegowski M, Flamand MN, Meyer KD. scDART-seq reveals distinct m6A signatures and mRNA methylation heterogeneity in single cells. Mol Cell. 2022 Feb 17;82(4):868-878.e10. Epub 2022 Jan 25 PubMed.
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