Epigenetic memory of drug exposure history controls neural stem cell quiescence in the adult brain

Masakazu Iwamoto,Taito Matsuda

Neural stem cells (NSCs) are the source of all neurons and glial cells (astrocytes and oligodendrocytes) in the central nervous system.The adult mammalian brain retains NSCs in the subgranular zone of the dentate gyrus in the hippocampus and ventricular subventricular zone lining the lateral ventricle (Olpe and Jessberger,2022).Adult NSCs in rodents are preserved throughout life and continuously produce new neurons that integrate into the pre-existing neuronal network.However,whether adult neurogenesis occurs in humans,especially in the hippocampus,remains yet to be proven (Olpe and Jessberger,2022).Adult neurogenesis in the hippocampus contributes to hippocampusdependent cognitive function (Gonçalves et al.,2016).Disruption of this neurogenesis is known to be associated with several brain disorders,such as age-dependent cognitive decline,major depressive disorders,and medial-temporal lobe epilepsy.Thus,elucidating the mechanisms underlying the regulation of NSC behavior and neurogenesis is important for developing therapeutic strategies to treat diseases related to impaired adult neurogenesis.

The process of hippocampal adult neurogenesis begins with Nestin-,Gfap-and Sox2-expressing quiescent NSCs,also called radial glia-like cells because of their morphology and ontogeny (Mira et al.,2010;Doi et al.,2021).Whether NSCs remain quiescent or enter an active state is determined by niche cells through direct cellcell contacts and secreted molecules.Bone morphogenetic proteins (BMPs),produced by granule neurons and NSCs themselves,are required to promote quiescence and long-term maintenance of NSCs through BMP-Smad signaling (Mira et al.,2010).BMP type I receptors that have been activated by BMPs phosphorylate the latent transcription factor Smad 1 and induce its nuclear translocation,resulting in the expression of Smad target factors,such as Id1-4,which can inhibit the function of proneural transcription factors,to decrease cell proliferation and induce the quiescent state (Mira et al.,2010).Notch signaling enables short-range intercellular communication between adjacent cells through the interaction with membrane-anchored ligands,Delta and Jagged.Among Notch family proteins,Notch2 has been shown to maintain NSC quiescence through the expression of Id4 (Zhang et al.,2019).The participation of neuronal networks in regulating adult NSCs has also been demonstrated.Among several interneuron cell types,parvalbuminexpressing interneurons in the hippocampal dentate region function as unique local circuit components that activate gamma-aminobutyric acid (GABA) signaling and maintain quiescence in adult brain cells.In contrast,diazepam binding inhibitor,a factor that dampens GABA activity,is known to induce NSC activation.GABAAreceptors are ion channel-type receptors that selectively allow Cl-to permeate through the binding of GABA.Diazepam binding inhibitor binds to the GABAAreceptor and negatively modulates GABAinduced currents.NSCs in the hippocampus autonomously express diazepam binding inhibitor,which negatively modulates GABA signaling to promote NSC proliferation (Bao and Song,2018).

Epigenetics involves changes in genome function that occur without a change in the DNA sequence.The ability of cells to stably retain and transmit their unique gene expression patterns to the daughter cells,which is referred to as epigenetic memory,is encoded by epigenetic marks and the associated epigenetic memory factors.Epigenetic memory is central to the maintenance of cell identities established by the response of gene expression states to developmental and environmental signaling and plays an important role in adaptation to the environment (Steffen and Ringrose,2014;Naik and Fuchs,2022).

In mammals,epigenetic memory is well studied as the mechanism of inflammatory memory that manifests as an adaptation to a second inflammatory,metabolic,or microbial stimulus.Interestingly,inflammatory genes,e.g.,interleukin-6,showed tolerizing behavior,i.e.,loss of the active chromatin mark on their promoter and weakened transcription after a second lipopolysaccharide exposure.In contrast,antimicrobial genes showed priming behavior,increasing their expression upon lipopolysaccharide restimulation.This facilitated transcriptional response was attributed to the rapid mobilization of RNA polymerase II to the promoters of primed genes (Naik and Fuchs,2022).Following an initial inflammatory or microbial trigger,cells alter histone and chromatin accessibility to activate the transcription of inflammatory,antibacterial,and stress-related genes.Most of these genes quickly return to their baseline epigenetic state upon withdrawal of stimulation.In contrast,the resolution of H3K4me1 marks in enhancers and/or H3K4me3 marks in proximal promoters is slow or absent in some defined gene loci.Chromatin also remains accessible at these relevant loci,allowing rapid recruitment of RNA polymerase II and transcriptional activation at secondary triggers.The proliferation and differentiation of stem cells,including NSCs,are well known to be regulated by epigenetics,and therefore it is reasonable to assume that epigenetic memory may play a significant role in controlling the behavior of stem cells.

Several human epidemiological studies have demonstrated that repeated early-life exposure to anesthesia,a positive modulator of GABAAreceptors,is associated with adverse neurobehavioral outcomes in later life,such as a higher risk of learning disability and attention deficit hyperactivity disorder.We have recently shown that early-life exposure to midazolam (MDZ),a widely used drug in pediatric anesthesia,persistently alters chromatin accessibility and the expression of quiescence-associated genes in NSCs in the mouse hippocampus (Doi et al.,2021).We administered MDZ intraperitoneally to postnatal day 7 (P7) mice daily for 3 consecutive days (MDZ mice) and found that repeated earlylife MDZ exposure induced an abnormal increase of quiescent NSCs at P10.Interestingly,the proportion of active NSCs among total NSCs was lower in the dentate gyrus of 8-week-old MDZ mice,indicating that MDZ-induced dormancy of NSCs is long-lasting,although the anesthetic effect of MDZ lasts only 2-3 hours after administration.We also found that the lasting effects of MDZ on NSC dormancy led to a persistent decline in hippocampal neurogenesis and impaired hippocampus-dependent memory function in adult MDZ mice.

To elucidate the molecular mechanisms by which early MDZ exposure causes NSCs to form longterm quiescent memories,we performed RNA sequencing and Assay for Transposase-Accessible Chromatin sequencing of hippocampal NSCs at P10 and 8 weeks old with or without early-life MDZ exposure.We observed that the chromatin status in the vicinity of quiescence-associated genes in NSCs changed continuously from early postnatal to adult stages,resulting in a longterm upregulation of the quiescence-related genes.Furthermore,Egr1,a transcription factor known to recruit chromatin remodeling factors,was transiently upregulated in hippocampal NSCs in P10 MDZ-exposure mice.Forced expression of Egr1 caused NSC quiescence by upregulating quiescence-associated genes,such asNotch2andId4,suggesting that at least a fraction of the MDZ-induced gene expression changes were mediated through Egr1 expression.When we checked chromatin accessibility,Egr1-occupied loci were located in accessible chromatin regions,and chromatin accessibility at these loci,including theNotch2locus,was persistently higher,from P10 to 8 weeks old,in NSCs of MDZ mice than in Ctrl mice.Integrative analysis of Egr1 chromatin immunoprecipitation sequencing data from NSCs and public chromatin immunoprecipitation sequencing datasets suggested that Egr1 cooccupies with chromatin remodeling factors Chd8 and Smarca4,which increases chromatin accessibility.These findings suggest that Egr1 acts in concert with chromatin remodeling proteins (Chd8 and Smarca4) to cause epigenetic memory formation (quiescent memory) with the protracted increment of chromatin accessibility around quiescent gene-associated loci,resulting in longterm NSC dormancy (Figure 1).

Figure 1｜Mechanism of quiescent memory formation in hippocampal NSCs and adverse neurobehavioral outcomes in later life by early life MDZ-exposure.

Epigenetic memory of past experiences has been reported in the regulation of other tissuespecific stem cells.In naive muscle stem cells,regulatory sequences associated with injuryinduced activation are highly methylated.Many of these sites become demethylated following injury,thereby changing the expression of genes involved in myogenesis.These sites remain in this state as an epigenetic memory many months after the injury,representing a molecular memory of previous physiological events (Michaeli et al.,2022).Epidermal stem cells remember past acute inflammatory events by bookmarking,through epigenetic modifications,the chromatin accessibility of key transcription factors required for regeneration (Naik et al.,2017).This enables the subsequent faster transcription of genes necessary for tissue repair,leading to faster wound healing (Naik et al.,2017).Thus,epigenetic memory has been shown to form in tissue stem cells under various conditions,including tissue injury and drug exposure.Elucidating whether there is specificity in epigenetic memory generation,why stem cells tend to store and evoke memories,and how long they remember is key to understanding how tissues continually cope with a myriad of external and internal stimuli.

Another critical issue is determining whether it is possible to erase epigenetic memories once formed since erasing epigenetic memories that negatively affect stem cell behavior may lead to the reactivation of stem cells.We have found that exercise in the form of voluntary wheel running,a well-known positive neurogenic stimulus,largely normalized the disturbed gene expression patterns in NSCs of MDZ mice,leading to the activation of dormant NSCs and improved impaired hippocampus-dependent memory function.Although further investigation is required to reveal whether voluntary exercise induces chromatin remodeling and releases epigenetic memory once formed,control of NSCs and subsequent neurogenesis holds promise as a potential therapeutic strategy to treat brain diseases.Voluntary exercise has also been shown to positively affect other tissue stem cells,such as improving the function of aged muscle stem cells and promoting muscle repair in aged mice (Brett et al.,2020).Advances in our understanding of the processes underlying epigenetic memory formation and how to unlock them may establish therapeutic strategies using tissue-specific stem cells,such as for skin and muscle disorders,in addition to neurological disorders.

This work was supported by a Grant-in-Aid for Scientific Research (B) JP21H02808 (to TM) and JST SPRING JPMJSP2136 (to MI).

Masakazu Iwamoto,Taito Matsuda*

Department of Stem Cell Biology and Medicine,Graduate School of Medical Sciences,Kyushu University,Fukuoka,Japan

*Correspondence to:Taito Matsuda,PhD,matsuda.taito.344@m.kyushu-u.ac.jp.https://orcid.org/0000-0002-9859-5967(Taito Matsuda)

Date of submission:April 28,2023

Date of decision:June 25,2023

Date of acceptance:July 7,2023

Date of web publication:August 14,2023

https://doi.org/10.4103/1673-5374.382240

How to cite this article:Iwamoto M,Matsuda T (2024) Epigenetic memory of drug exposure history controls neural stem cell quiescence in the adult brain.Neural Regen Res 19(4):711-712.

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