m6A/YTHDF2-mediated mRNA decay targets TGF-β signaling to suppress the quiescence acquisition of early postnatal mouse hippocampal NSCs

Zhao T., Hong Y., Yan B., Huang S., Ming G., Song H.

AbstractQuiescence, a hallmark of adult neural stem cells (NSCs), is required for maintaining the NSC pool to support life-long continuous neurogenesis in the adult dentate gyrus (DG). Whether long-lasting epigenetic modifications maintain NSC quiescence over the long term in the adult DG is not well-understood. Here we show that mice with haploinsufficiency of Setd1a, a schizophrenia risk gene encoding a histone H3K4 methyltransferase, develop an enlarged DG with more dentate granule cells after young adulthood. Deletion of Setd1a specifically in quiescent NSCs in the adult DG promotes their activation and neurogenesis, which is countered by inhibition of the histone demethylase LSD1. Mechanistically, RNA-sequencing and CUT & RUN analyses of cultured quiescent adult NSCs reveal Setd1a deletion-induced transcriptional changes and many Setd1a targets, among which down-regulation of Bhlhe40 promotes quiescent NSC activation in the adult DG in vivo. Together, our study reveals a Setd1a-dependent epigenetic mechanism that sustains NSC quiescence in the adult DG.

Jimenez-Cyrus D., Adusumilli V.S., Stempel M.H., Maday S., Ming G., Song H., Bond A.M.

Quiescent adult neural stem cells (NSCs) in the mammalian brain arise from proliferating NSCs during development. Beyond acquisition of quiescence, an adult NSC hallmark, little is known about the process, milestones, and mechanisms underlying the transition of developmental NSCs to an adult NSC state. Here, we performed targeted single-cell RNA-seq analysis to reveal the molecular cascade underlying NSC development in the early postnatal mouse dentate gyrus. We identified two sequential steps, first a transition to quiescence followed by further maturation, each of which involved distinct changes in metabolic gene expression. Direct metabolic analysis uncovered distinct milestones, including an autophagy burst before NSC quiescence acquisition and cellular reactive oxygen species level elevation along NSC maturation. Functionally, autophagy is important for the NSC transition to quiescence during early postnatal development. Together, our study reveals a multi-step process with defined milestones underlying establishment of the adult NSC pool in the mammalian brain.

Zhang F., Ignatova V.V., Ming G., Song H.

Various chemical modifications of all RNA transcripts, or epitranscriptomics, have emerged as crucial regulators of RNA metabolism, attracting significant interest from both basic and clinical researchers due to their diverse functions in biological processes and immense clinical potential as highlighted by the recent profound success of RNA modifications in improving COVID-19 mRNA vaccines. Rapid accumulation of evidence underscores the critical involvement of various RNA modifications in governing normal neural development and brain functions as well as pathogenesis of brain disorders. Here we provide an overview of RNA modifications and recent advancements in epitranscriptomic studies utilizing animal models to elucidate important roles of RNA modifications in regulating mammalian neurogenesis, gliogenesis, synaptic formation, and brain function. Moreover, we emphasize the pivotal involvement of RNA modifications and their regulators in the pathogenesis of various human brain disorders, encompassing neurodevelopmental disorders, brain tumors, psychiatric and neurodegenerative disorders. Furthermore, we discuss potential translational opportunities afforded by RNA modifications in combatting brain disorders, including their use as biomarkers, in the development of drugs or gene therapies targeting epitranscriptomic pathways, and in applications for mRNA-based vaccines and therapies. We also address current limitations and challenges hindering the widespread clinical application of epitranscriptomic research, along with the improvements necessary for future progress.

Calatayud-Baselga I., Casares-Crespo L., Franch-Ibáñez C., Guijarro-Nuez J., Sanz P., Mira H.

AbstractNeurogenesis in the adult mammalian brain relies on the lifelong persistence of quiescent neural stem cell (NSC) reservoirs. Little is known about the mechanisms that lead to the initial establishment of quiescence, the main hallmark of adult stem cells, during development. Here we show that protein aggregates and autophagy machinery components accumulate in developmental radial glia-like NSCs as they enter quiescence and that pharmacological or genetic blockade of autophagy disrupts quiescence acquisition and maintenance. Conversely, increasing autophagy through AMPK/ULK1 activation instructs the acquisition of the quiescent state without affecting BMP signaling, a gatekeeper of NSC quiescence during adulthood. Selective ablation of Atg7, a critical gene for autophagosome formation, in radial glia-like NSCs at early and late postnatal stages compromises the initial acquisition and maintenance of quiescence during the formation of the hippocampal dentate gyrus NSC niche. Therefore, we demonstrate that autophagy is cell-intrinsically required to establish NSC quiescence during hippocampal development. Our results uncover an important role of autophagy in the transition of developmental NSCs into their dormant adult form, paving the way for studies directed at further understanding the mechanisms of stem cell niche formation and maintenance in the mammalian brain.

Massagué J., Sheppard D.

The TGF-β regulatory system plays crucial roles in the preservation of organismal integrity. TGF-β signaling controls metazoan embryo development, tissue homeostasis, and injury repair through coordinated effects on cell proliferation, phenotypic plasticity, migration, metabolic adaptation, and immune surveillance of multiple cell types in shared ecosystems. Defects of TGF-β signaling, particularly in epithelial cells, tissue fibroblasts, and immune cells, disrupt immune tolerance, promote inflammation, underlie the pathogenesis of fibrosis and cancer, and contribute to the resistance of these diseases to treatment. Here, we review how TGF-β coordinates multicellular response programs in health and disease and how this knowledge can be leveraged to develop treatments for diseases of the TGF-β system.

Zhang F., Yoon K., Kim N., Ming G., Song H.

Quiescence is a hallmark of adult neural stem cells (NSCs) in the mammalian brain, and establishment and maintenance of quiescence is essential for life-long continuous neurogenesis. How NSCs in the dentate gyrus (DG) of the hippocampus acquire their quiescence during early postnatal stages and continuously maintain quiescence in adulthood is poorly understood. Here, we show that Hopx-CreERT2-mediated conditional deletion of Nkcc1, which encodes a chloride importer, in mouse DG NSCs impairs both their quiescence acquisition at early postnatal stages and quiescence maintenance in adulthood. Furthermore, PV-CreERT2-mediated deletion of Nkcc1 in PV interneurons in the adult mouse brain leads to activation of quiescent DG NSCs, resulting in an expanded NSC pool. Consistently, pharmacological inhibition of NKCC1 promotes NSC proliferation in both early postnatal and adult mouse DG. Together, our study reveals both cell-autonomous and non-cell-autonomous roles of NKCC1 in regulating the acquisition and maintenance of NSC quiescence in the mammalian hippocampus.

Lin Z., Zhao S., Li X., Miao Z., Cao J., Chen Y., Shi Z., Zhang J., Wang D., Chen S., Wang L., Gu A., Chen F., Yang T., Sun K., et. al.

Genetic information is generally transferred from RNA to protein according to the classic “Central Dogma”. Here, we made a striking discovery that post-translational modification of a protein specifically regulates the editing of its own mRNA. We show that S-nitrosylation of cathepsin B (CTSB) exclusively alters the adenosine-to-inosine (A-to-I) editing of its own mRNA. Mechanistically, CTSB S-nitrosylation promotes the dephosphorylation and nuclear translocation of ADD1, leading to the recruitment of MATR3 and ADAR1 to CTSB mRNA. ADAR1-mediated A-to-I RNA editing enables the binding of HuR to CTSB mRNA, resulting in increased CTSB mRNA stability and subsequently higher steady-state levels of CTSB protein. Together, we uncovered a unique feedforward mechanism of protein expression regulation mediated by the ADD1/MATR3/ADAR1 regulatory axis. Our study demonstrates a novel reverse flow of information from the post-translational modification of a protein back to the post-transcriptional regulation of its own mRNA precursor. We coined this process as “Protein-directed EDiting of its Own mRNA by ADAR1 (PEDORA)” and suggest that this constitutes an additional layer of protein expression control. “PEDORA” could represent a currently hidden mechanism in eukaryotic gene expression regulation.

Guo Z., Liu X., Zhao S., Sun F., Ren W., Ma M.

Zhang F., Yoon K., Zhang D.Y., Kim N., Ming G., Song H.

Increasing evidence implicates the critical roles of various epitranscriptomic RNA modifications in different biological processes. Methyltransferase METTL8 installs 3-methylcytosine (m3C) modification of mitochondrial tRNAs in vitro; however, its role in intact biological systems is unknown. Here, we show that Mettl8 is localized in mitochondria and installs m3C specifically on mitochondrial tRNAThr/Ser(UCN) in mouse embryonic cortical neural stem cells. At molecular and cellular levels, Mettl8 deletion in cortical neural stem cells leads to reduced mitochondrial protein translation and attenuated respiration activity. At the functional level, conditional Mettl8 deletion in mice results in impaired embryonic cortical neural stem cell maintenance in vivo, which can be rescued by pharmacologically enhancing mitochondrial functions. Similarly, METTL8 promotes mitochondrial protein expression and neural stem cell maintenance in human forebrain cortical organoids. Together, our study reveals a conserved epitranscriptomic mechanism of Mettl8 and mitochondrial tRNA m3C modification in maintaining embryonic cortical neural stem cells in mice and humans.

He P.C., Wei J., Dou X., Harada B.T., Zhang Z., Ge R., Liu C., Zhang L., Yu X., Wang S., Lyu R., Zou Z., Chen M., He C.

N 6 -methyladenosine (m 6 A) is the most abundant messenger RNA (mRNA) modification and plays crucial roles in diverse physiological processes. Using a massively parallel assay for m 6 A (MPm 6 A), we discover that m 6 A specificity is globally regulated by suppressors that prevent m 6 A deposition in unmethylated transcriptome regions. We identify exon junction complexes (EJCs) as m 6 A suppressors that protect exon junction–proximal RNA within coding sequences from methylation and regulate mRNA stability through m 6 A suppression. EJC suppression of m 6 A underlies multiple global characteristics of mRNA m 6 A specificity, with the local range of EJC protection sufficient to suppress m 6 A deposition in average-length internal exons but not in long internal and terminal exons. EJC-suppressed methylation sites colocalize with EJC-suppressed splice sites, which suggests that exon architecture broadly determines local mRNA accessibility to regulatory complexes.

Micaelli M., Dalle Vedove A., Cerofolini L., Vigna J., Sighel D., Zaccara S., Bonomo I., Poulentzas G., Rosatti E.F., Cazzanelli G., Alunno L., Belli R., Peroni D., Dassi E., Murakami S., et. al.

YTHDF proteins bind the N6-methyladenosine (m6A)-modified mRNAs, influencing their processing, stability, and translation. Therefore, the members of this protein family play crucial roles in gene regulation and several physiological and pathophysiological conditions. YTHDF proteins contain a hydrophobic pocket that accommodates the m6A embedded in the RRACH consensus sequence on mRNAs. We exploited the presence of this cage to set up an m6A-competitive assay and performed a high-throughput screen aimed at identifying ligands binding in the m6A pocket. We report the organoselenium compound ebselen as the first-in-class inhibitor of the YTHDF m6A-binding domain. Ebselen, whose interaction with YTHDF proteins was validated via orthogonal assays, cannot discriminate between the binding domains of the three YTHDF paralogs but can disrupt the interaction of the YTHDF m6A domain with the m6A-decorated mRNA targets. X-ray, mass spectrometry, and NMR studies indicate that in YTHDF1 ebselen binds close to the m6A cage, covalently to the Cys412 cysteine, or interacts reversibly depending on the reducing environment. We also showed that ebselen engages YTHDF proteins within cells, interfering with their mRNA binding. Finally, we produced a series of ebselen structural analogs that can interact with the YTHDF m6A domain, proving that ebselen expansion is amenable for developing new inhibitors. Our work demonstrates the feasibility of drugging the YTH domain in YTHDF proteins and opens new avenues for the development of disruptors of m6A recognition.

Zhou Y., Su Y., Li S., Kennedy B.C., Zhang D.Y., Bond A.M., Sun Y., Jacob F., Lu L., Hu P., Viaene A.N., Helbig I., Kessler S.K., Lucas T., Salinas R.D., et. al.

Immature dentate granule cells (imGCs) arising from adult hippocampal neurogenesis contribute to plasticity and unique brain functions in rodents1,2 and are dysregulated in multiple human neurological disorders3–5. Little is known about the molecular characteristics of adult human hippocampal imGCs, and even their existence is under debate1,6–8. Here we performed single-nucleus RNA sequencing aided by a validated machine learning-based analytic approach to identify imGCs and quantify their abundance in the human hippocampus at different stages across the lifespan. We identified common molecular hallmarks of human imGCs across the lifespan and observed age-dependent transcriptional dynamics in human imGCs that suggest changes in cellular functionality, niche interactions and disease relevance, that differ from those in mice9. We also found a decreased number of imGCs with altered gene expression in Alzheimer's disease. Finally, we demonstrated the capacity for neurogenesis in the adult human hippocampus with the presence of rare dentate granule cell fate-specific proliferating neural progenitors and with cultured surgical specimens. Together, our findings suggest the presence of a substantial number of imGCs in the adult human hippocampus via low-frequency de novo generation and protracted maturation, and our study reveals their molecular properties across the lifespan and in Alzheimer's disease. Single-nucleus RNA-sequencing analysis supports the presence of immature dentate granule cells throughout the human lifespan and shows that these cells are reduced in number and dysregulated in Alzheimer's disease.

Hagemann-Jensen M., Ziegenhain C., Sandberg R.

Current single-cell RNA sequencing (scRNA-seq) methods with high cellular throughputs sacrifice full-transcript coverage and often sensitivity. Here we describe Smart-seq3xpress, which miniaturizes and streamlines the Smart-seq3 protocol to substantially reduce reagent use and increase cellular throughput. Smart-seq3xpress analysis of peripheral blood mononuclear cells resulted in a granular atlas complete with common and rare cell types. Compared with droplet-based single-cell RNA sequencing that sequences RNA ends, the additional full-transcript coverage revealed cell-type-associated isoform variation. Smart-seq3xpress enables single-cell RNA sequencing from full transcripts at high throughput.

Terreros-Roncal J., Moreno-Jiménez E.P., Flor-García M., Rodríguez-Moreno C.B., Trinchero M.F., Cafini F., Rábano A., Llorens-Martín M.

Disease and hippocampal dysfunction Impaired function of the brain’s hippocampus can underpin psychiatric symptoms and cognitive impairment. Looking at postmortem brain samples from patients affected by any of several neurodegenerative disorders, Terreros-Roncal et al . investigated whether adult neurogenesis was disrupted (see the Perspective by Gage). Indeed, functions of the neurogenic niche shifted and the cells produced were abnormal in shape and differentiation. The neuronal plasticity characteristic of the hippocampus may make it especially susceptible to the ravages of neurodegenerative disease. —PJH

Morais P., Adachi H., Yu Y.

The current COVID-19 pandemic is a massive source of global disruption, having led so far to two hundred and fifty million COVID-19 cases and almost five million deaths worldwide. It was recognized in the beginning that only an effective vaccine could lead to a way out of the pandemic, and therefore the race for the COVID-19 vaccine started immediately, boosted by the availability of the viral sequence data. Two novel vaccine platforms, based on mRNA technology, were developed in 2020 by Pfizer-BioNTech and Moderna Therapeutics (comirnaty® and spikevax®, respectively), and were the first ones presenting efficacies higher than 90%. Both consisted of N1-methyl-pseudouridine-modified mRNA encoding the SARS-COVID-19 Spike protein and were delivered with a lipid nanoparticle (LNP) formulation. Because the delivery problem of ribonucleic acids had been known for decades, the success of LNPs was quickly hailed by many as the unsung hero of COVID-19 mRNA vaccines. However, the clinical trial efficacy results of the Curevac mRNA vaccine (CVnCoV) suggested that the delivery system was not the only key to the success. CVnCoV consisted of an unmodified mRNA (encoding the same spike protein as Moderna and Pfizer-BioNTech’s mRNA vaccines) and was formulated with the same LNP as Pfizer-BioNTech’s vaccine (Acuitas ALC-0315). However, its efficacy was only 48%. This striking difference in efficacy could be attributed to the presence of a critical RNA modification (N1-methyl-pseudouridine) in the Pfizer-BioNTech and Moderna’s mRNA vaccines (but not in CVnCoV). Here we highlight the features of N1-methyl-pseudouridine and its contributions to mRNA vaccines.

Chen T., Ye W., Gao S., Li Y., Luan J., Lv X., Wang S.

Liang Z., Jin N., Guo W.

Abstract Adult neurogenesis is a unique cellular process of the ongoing generation of new neurons throughout life, which primarily occurs in the subgranular zone (SGZ) of the dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricle. In the adult DG, newly generated granule cells from neural stem cells (NSCs) integrate into existing neural circuits, significantly contributing to cognitive functions, particularly learning and memory. Recently, more and more studies have shown that rather than being a homogeneous population of identical cells, adult NSCs are composed of multiple subpopulations that differ in their morphology and function. In this study, we provide an overview of the origin, regional characteristics, prototypical morphology, and molecular factors that contribute to NSC heterogeneity. In particular, we discuss the molecular mechanisms underlying the balance between activation and quiescence of NSCs. In summary, this review highlights that deciphering NSC heterogeneity in the adult brain is a challenging but critical step in advancing our understanding of tissue-specific stem cells and the process of neurogenesis in the adult brain.