Extra-coding RNAs regulate neuronal DNA methylation dynamics

Savell, Katherine E.; Gallus, Nancy; Simon, Rhiana C.; Brown, Jennifer; Revanna, Jasmin S.; Osborn, Mary Katherine; Song, Esther Y.; O’Malley, John J.; Stackhouse, Christian T.; Norvil, Allison B; Gowher, Humaira; Sweatt, J. David; Day, Jeremy J.

doi:10.1038/ncomms12091

Cited by 55 publications

(92 citation statements)

References 56 publications

(79 reference statements)

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“…However, drugs of abuse target many distinct receptor classes in a variety of neuronal and non-neuronal cell types (20,21), and these complex drug actions make identification of DA-induced gene expression programs difficult using in vivo models. Therefore, we took advantage of a well-studied and controllable rat primary striatal neuron culture system (35,36) to enable comprehensive and specific identification of DA-dependent gene expression programs in MSNs. Using this system, we first identified a core signature of the transcriptional response to DA receptor activation by performing deep RNA-seq on bulk striatal neuronal cultures treated with 1µM DA for 1hr, a treatment that closely models concentrations and temporal duration of DA increases found in vivo after acute cocaine exposure (2,3,6).…”

Section: Genesmentioning

confidence: 99%

“…The CREB inhibitor 666-15 (1µM; Tocris, 5661), also called 3-(3-Aminopropoxy)-N- Neuronal Cell Cultures. Primary rat neuronal cultures were generated from E18 rat striatal tissue as described previously (35,36). Briefly, cell culture plates (Denville Scientific Inc.) were coated overnight with poly-L-lysine (Sigma-Aldrich; 50 µg/ ml), supplemented with 7.5 µg/mL laminin (Sigma-Aldrich), and rinsed with diH 2 O. Microelectrode arrays (MEAs; Axion Biosystems) were coated with polyethyleneimine (Sigma-Aldrich).…”

Section: Drugsmentioning

confidence: 99%

“…Total RNA was extracted (RNAeasy kit, Qiagen) and reverse-transcribed (iScript cDNA Synthesis Kit, Bio-Rad). cDNA was subject to RT-qPCR for genes of interest, as described previously (36). A list of PCR primer sequences is provided in Supplemental Table 12.…”

Section: Rna Extraction and Rt-qpcrmentioning

confidence: 99%

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A dopamine-induced gene expression signature regulates neuronal function and cocaine response

Savell

Zipperly

Tuscher

et al. 2019

Preprint

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Drug addiction is a worldwide health problem, with overdose rates of both psychostimulants and opioids currently on the rise in many developed countries. Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. However, even with well-studied drugs such as cocaine, druginduced transcriptional responses remain poorly understood due to the cellular heterogeneity of the NAc and complex drug actions via multiple neurotransmitter systems. Here, we leveraged highthroughput single-nucleus RNA-sequencing to create a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type-specific responses to acute cocaine experience in a rat model system. Using this transcriptional map, we identified specific neuronal subpopulations that are activated by cocaine, and defined an immediate early gene expression program that is upregulated following cocaine experience in vivo and dopamine (DA) receptor activation in vitro. To characterize the neuronal response to this DA-mediated gene expression signature, we engineered a large-scale CRISPR/dCas9 activation strategy to recreate this program. Multiplexed induction of this gene program initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Taken together, these results define the genome-wide transcriptional response to cocaine with cellular precision, and demonstrate that drug-responsive gene programs are sufficient to initiate both physiological and behavioral adaptations to drugs of abuse.NEARLY 5 MILLION Americans reported cocaine use in 2017, and recent increases in cocaine-related drug overdoses present significant public health challenges (1). A hallmark trait of drugs of abuse is the acute elevation of dopamine (DA) in the nucleus accumbens (NAc), a central integrator of the reward circuit (2-4). Abused drugs produce increases in DA that are greater in both concentration and duration than natural rewards (2,5,6), and this signaling is hypothesized to underlie maladaptive reinforcement after repeated drug use (7). Exposure to drugs of abuse results in significant transcriptional and epigenetic reorganization in the NAc (8-13), initiating synaptic and behavioral plasticity associated with the transition to drug addiction (7,14,15). However, even with well-studied drugs such as cocaine, drug-induced transcriptional responses remain poorly understood. This is in part due to the cellular heterogeneity of the NAc, which is a diverse structure containing multiple neuronal and non-neuronal subpopulations and complex neuronal circuitry. Additionally, many drugs of abuse engage multiple neurotransmitter systems in the NAc (16-22), and the specific contributions of DA-dependent transcriptional programs to neuronal physiology and behavior is not clear. Further, although drug experience leads to large scale transcriptional changes in the NAc, previou...

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Section: Genesmentioning

confidence: 99%

Section: Drugsmentioning

confidence: 99%

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A dopamine-induced gene expression signature regulates neuronal function and cocaine response

Savell

Zipperly

Tuscher

et al. 2019

Preprint

Self Cite

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“…76 Recent research suggests that this may occur through extracoding RNAs (ecRNAs) a form of non-coding RNAs, which are transcribed in parallel with IEGs, are induced by neuronal activity, and bind to de novo DNMTs to methylate their corresponding gene promoters. 84 Regardless of the exact mechanisms involved, evidence strongly points toward a specific role of DNA methylation in regulating synaptic plasticity-related transcripts. [85][86][87] Taken together, work in learning and memory provides a model of activity-dependent epigenetic mechanisms that may also be impacted by altered GR signaling and stress-response in MDD, leading to both immediate and long-lasting impairments in cellular functioning and cognition.…”

Section: Activity-dependent Chromatin Opening Via Histone Modificationmentioning

confidence: 99%

A role for activity‐dependent epigenetics in the development and treatment of major depressive disorder

Nagy

Vaillancourt

Turecki

2018

Genes Brain and Behavior

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Chronic stressors, during developmental sensitive periods and beyond, contribute to the risk of developing psychiatric conditions, including major depressive disorder (MDD). Epigenetic mechanisms including DNA methylation and histone modifications, at key stress response and neurotrophin genes, are increasingly implicated in mediating this risk. Although the exact mechanisms through which stressful environmental stimuli alter the epigenome are still unclear, research from the learning and memory fields indicates that epigenomic marks can be altered, at least in part, through calcium-dependent signaling cascades in direct response to neuronal activity. In this review, we highlight key findings from the stress, MDD, and learning and memory fields to propose a model where stress regulates downstream cellular functioning through activity-dependent epigenetic changes. Furthermore, we suggest that both typical and novel antidepressant treatments may exert positive influence through similar, activity-dependent pathways.

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“…Neuronal Cell Cultures. Primary rat neuronal cultures were generated from embryonic day 18 (E18) rat cortical tissue, as described previously [19][20][21] . Briefly, cell culture plates (Denville Scientific Inc.) were coated overnight with poly-L-lysine (Sigma-Aldrich; 50 µg/ml) and rinsed with diH 2 O. Dissected cortical tissue was incubated with papain (Worthington LK003178) for 25 min at 37°C.…”

Section: Animalsmentioning

confidence: 99%

Blue light-induced gene expression alterations in cultured neurons are the result of phototoxic interactions with neuronal culture media

Duke

Savell

Phillips

et al. 2019

Preprint

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Blue waveform light is used as an optical actuator in numerous optogenetic technologies employed in neuronal systems. However, the potential side effects of blue waveform light in neurons has not been thoroughly explored, and recent reports suggest that neuronal exposure to blue light can induce transcriptional alterations in vitro and in vivo. Here, we examined the effects of blue waveform light in cultured primary rat cortical neurons. Exposure to blue light (470nm) resulted in upregulation of several immediate early genes (IEGs) traditionally used as markers of neuronal activity, including Fos and Fosb, but did not alter the expression of circadian clock genes Bmal1, Cry1, Cry2, Clock, or Per2. IEG expression was increased following 4 hours of 5% duty cycle light exposure, and IEG induction was not dependent on light pulse width. Elevated levels of blue light exposure induced a loss of cell viability in vitro, suggestive of overt phototoxicity. Changes in gene expression induced by blue waveform light were prevented when neurons were cultured in a photoinert media supplemented with a photostable neuronal supplement instead of commonly utilized neuronal culture media and supplements. Together, these findings suggest that light-induced gene expression alterations observed in vitro stem from a phototoxic interaction between commonly used media and neurons, and offer a solution to prevent this toxicity when using photoactivatable technology in vitro.

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Extra-coding RNAs regulate neuronal DNA methylation dynamics

Cited by 55 publications

References 56 publications

A dopamine-induced gene expression signature regulates neuronal function and cocaine response

A dopamine-induced gene expression signature regulates neuronal function and cocaine response

A role for activity‐dependent epigenetics in the development and treatment of major depressive disorder

Blue light-induced gene expression alterations in cultured neurons are the result of phototoxic interactions with neuronal culture media

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