O-GlcNAc and EZH2-mediated epigenetic regulation of gene expression during consolidation of fear memories.

O-GlcNAcylation of serine/threonine residues on target proteins occurs dynamically in postmitotic neurons of the hippocampus and may serve to control both the stability and activity of target proteins. Remarkably, the addition and removal of the O-GlcNAc posttranslational modifications are catalyzed by a pair of enzymes, the O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). More than thousands of proteins are modified by O-GlcNAcylation including epigenetic modifying enzymes. A critical target of OGT is the polycomb repressive complex 2 (PRC2) containing the histone lysine methyltransferase EZH2 that mediates trimethylation of lysine 27 on histone H3 (H3K27me3). However, whether OGT and PRC2 activity in the hippocampus couple to regulate gene transcription mechanisms during memory consolidation remains unknown. Here, we found increases in OGT expression and global O-GlcNAcylation levels in dorsal area CA1 of the hippocampus during memory consolidation. Additionally, we observed that OGT exerts control over epigenetic regulation via EZH2-H3K27me3 during memory consolidation. Blocking O-GlcNAc signaling via RNAi within dorsal area CA1 led to the global and site-specific loss of activity-dependent epigenetic plasticity at genes regulated by H3K27me3 and impairment of hippocampus-dependent memory. Together, these findings illustrate a unique epigenetic role of OGT via regulation of histone methylation mediated by EZH2 during memory consolidation of fear conditioned memories.

Human and rodent temporal lobe epilepsy is characterized by changes in O-GlcNAc homeostasis that can be reversed to dampen epileptiform activity.

Temporal Lobe Epilepsy (TLE) is frequently associated with changes in protein composition and post-translational modifications (PTM) that exacerbate the disorder. O-linked-ß-N-acetyl glucosamine (O-GlcNAc) is a PTM occurring at serine/threonine residues that is derived from and closely associated with metabolic substrates. The enzymes O-GlcNActransferase (OGT) and O-GlcNAcase (OGA) mediate the addition and removal, respectively, of the O-GlcNAc modification. The goal of this study was to characterize OGT/OGA and protein O-GlcNAcylation in the epileptic hippocampus and to determine and whether direct manipulation of these proteins and PTM's alter epileptiform activity. We observed reduced global and protein specific O-GlcNAcylation and OGT expression in the kainate rat model of TLE and in human TLE hippocampal tissue. Inhibiting OGA with Thiamet-G elevated protein O-GlcNAcylation, and decreased both seizure duration and epileptic spike events, suggesting that OGA may be a therapeutic target for seizure control. These findings suggest that loss of O-GlcNAc homeostasis in the kainate model and in human TLE can be reversed via targeting of O-GlcNAc related pathways.

Amygdalar expression of the microRNA miR-101a and its target Ezh2 contribute to rodent anxiety-like behaviour.

A greater understanding of neural mechanisms contributing to anxiety is needed in order to develop better therapeutic interventions. This study interrogates a novel molecular mechanism that shapes anxiety-like behaviour, demonstrating that the microRNA miR-101a-3p and its target, enhancer of zeste homolog 2 (Ezh2) in the amygdala, contribute to rodent anxiety-like behaviour. We utilized rats that were selectively bred for differences in emotionality and stress reactivity, showing that high-novelty-responding (HR) rats, which display low trait anxiety, have lower miR-101a-3p levels in the amygdala compared to low-novelty-responding (LR) rats that characteristically display high trait anxiety. To determine whether there is a causal relationship between amygdalar miR-101a-3p and anxiety behaviour, we used a viral approach to overexpress miR-101a-3p in the amygdala of HR rats and test whether it would increase their typically low levels of anxiety-like behaviour. We found that increasing miR-101a-3p in the amygdala increased HRs' anxiety-like behaviour in the open-field test and elevated plus maze. Viral-mediated miR-101a-3p overexpression also reduced expression of the histone methyltransferase Ezh2, which mediates gene silencing via trimethylation of histone 3 at lysine 27 (H3K27me3). Knockdown of Ezh2 with short-interfering RNA (siRNA) also increased HRs' anxiety-like behaviour, but to a lesser degree than miR-101a-3p overexpression. Overall, our data demonstrate that increasing miR-101a-3p expression in the amygdala increases anxiety-like behaviour and that this effect is at least partially mediated via repression of Ezh2. This work adds to the growing body of evidence implicating miRNAs and epigenetic regulation as molecular mediators of anxiety behaviour.

Dynamic association of epigenetic H3K4me3 and DNA 5hmC marks in the dorsal hippocampus and anterior cingulate cortex following reactivation of a fear memory.

Epigenetic mechanisms such as DNA methylation and histone methylation are critical regulators of gene transcription changes during memory consolidation. However, it is unknown how these epigenetic modifications coordinate control of gene expression following reactivation of a previously consolidated memory. Here, we found that retrieval of a recent contextual fear conditioned memory increased global levels of H3 lysine 4-trimethylation (H3K4me3) and DNA 5-hydroxymethylation (5hmC) in area CA1 of the dorsal hippocampus. Further experiments revealed increased levels of H3K4me3 and DNA 5hmC within a CpG-enriched coding region of the Npas4, but not c-fos, gene. Intriguingly, retrieval of a 30-day old memory increased H3K4me3 and DNA 5hmC levels at a CpG-enriched coding region of c-fos, but not Npas4, in the anterior cingulate cortex, suggesting that while these two epigenetic mechanisms co-occur following the retrieval of a recent or remote memory, their gene targets differ depending on the brain region. Additionally, we found that in vivo siRNA-mediated knockdown of the H3K4me3 methyltransferase Mll1 in CA1 abolished retrieval-induced increases in DNA 5hmC levels at the Npas4 gene, suggesting that H3K4me3 couples to DNA 5hmC mechanisms. Consistent with this, loss of Mll1 prevented retrieval-induced increases in Npas4 mRNA levels in CA1 and impaired fear memory. Collectively, these findings suggest an important link between histone methylation and DNA hydroxymethylation mechanisms in the epigenetic control of de novo gene transcription triggered by memory retrieval.

Ubiquitination of Histone H2B by Proteasome Subunit RPT6 Controls Histone Methylation Chromatin Dynamics During Memory Formation.

BACKGROUND: Posttranslational histone modifications play a critical role in the regulation of gene transcription underlying synaptic plasticity and memory formation. One such epigenetic change is histone ubiquitination, a process that is mediated by the ubiquitin-proteasome system in a manner similar to that by which proteins are normally targeted for degradation. However, histone ubiquitination mechanisms are poorly understood in the brain and in learning. In this article, we describe a new role for the ubiquitin-proteasome system in histone crosstalk, showing that learning-induced monoubiquitination of histone H2B (H2Bubi) is required for increases in the transcriptionally active H3 lysine 4 trimethylation (H3K4me3) mark at learning-related genes in the hippocampus. METHODS: Using a series of molecular, biochemical, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA-mediated knockdown and CRISPR (clustered regularly interspaced short palindromic repeats)-mediated upregulation of ubiquitin ligases, deubiquitinating enzymes and histone methyltransferases in the rat dorsal hippocampus during memory consolidation. RESULTS: We show that H2Bubi recruits H3K4me3 through a process that is dependent on the 19S proteasome subunit RPT6 and that a loss of H2Bubi in the hippocampus prevents learning-induced increases in H3K4me3, gene transcription, synaptic plasticity, and memory formation. Furthermore, we show that CRISPR-dCas9-mediated increases in H2Bubi promote H3K4me3 and memory formation under weak training conditions and that promoting histone methylation does not rescue memory impairments resulting from loss of H2Bubi. CONCLUSIONS: These results suggest that H2B ubiquitination regulates histone crosstalk in learning by way of nonproteolytic proteasome function, demonstrating a novel mechanism by which histone modifications are coordinated in response to learning.

The SETD6 Methyltransferase Plays an Essential Role in Hippocampus-Dependent Memory Formation.

BACKGROUND: Epigenetic mechanisms are critical for hippocampus-dependent memory formation. Building on previous studies that implicate the N-lysine methyltransferase SETD6 in the activation of nuclear factor-κB RELA (also known as transcription factor p65) as an epigenetic recruiter, we hypothesized that SETD6 is a key player in the epigenetic control of long-term memory. METHODS: Using a series of molecular, biochemical, imaging, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA-mediated knockdown of Setd6 in the rat dorsal hippocampus during memory consolidation. RESULTS: Our findings demonstrate that SETD6 is necessary for memory-related nuclear factor-κB RELA methylation at lysine 310 and associated increases in H3K9me2 (histone H3 lysine 9 dimethylation) in the dorsal hippocampus and that SETD6 knockdown interferes with memory consolidation, alters gene expression patterns, and disrupts spine morphology. CONCLUSIONS: Together, these findings suggest that SETD6 plays a critical role in memory formation and may act as an upstream initiator of H3K9me2 changes in the hippocampus during memory consolidation.

The Unexamined Calling.

Philosophy and philosophical assumptions are crucial to our work as physicians and investigators; however, training in philosophy remains poorly represented among medical students and their educators. In this brief essay, I argue the case that philosophical assumptions concerning knowledge and the scientific method are relevant for medical practice and research, concluding with a call for greater attention to philosophy in pre-medical and medical education.

Gamification: An Innovative Approach to Reinforce Clinical Knowledge for MD-PhD Students During Their PhD Research Years.

A longstanding challenge facing MD-PhD students and other dual-degree medical trainees is the loss of clinical knowledge that occurs during the non medical phases of training. Academic medical institutions nationwide have developed continued clinical training and exposure to maintain clinical competence; however, quantitative assessment of their usefulness remains largely unexplored. The current study therefore sought to both implement and optimize an online game platform to support MD-PhD students throughout their research training. Sixty three current MD-PhD students completing the PhD research phase of training were enrolled in an institutionally-developed online game platform for 2 preliminary and 4 competition rounds of 3-4 weeks each. During preliminary game rounds, we found that participation, though initially high, declined precipitously throughout the duration of each round, with 37 students participating to some extent. Daily reminders were implemented in subsequent rounds, which markedly improved player participation. Average participation in competition rounds exceeded 35% (23/63) active participants each round, with trending improvement in scores throughout the duration of PhD training. Both player participation and progress through the research phase of the MD-PhD program correlated positively with game performance and therefore knowledge retention and/or acquisition. Coupled with positive survey-based feedback from participants, our data therefore suggest that gamification is an effective tool for MD-PhD programs to combat loss of clinical knowledge during research training.

Rationalism, Empiricism, and Evidence-Based Medicine: A Call for a New Galenic Synthesis.

Thirty years after the rise of the evidence-based medicine (EBM) movement, formal training in philosophy remains poorly represented among medical students and their educators. In this paper, I argue that EBM’s reception in this context has resulted in a privileging of empiricism over rationalism in clinical reasoning with unintended consequences for medical practice. After a limited review of the history of medical epistemology, I argue that a solution to this problem can be found in the method of the 2nd-century Roman physician Galen, who brought empiricism and rationalism together in a synthesis anticipating the scientific method. Next, I review several of the problems that have been identified as resulting from a staunch commitment to empiricism in medical practice. Finally, I conclude that greater epistemological awareness in the medical community would precipitate a Galenic shift toward a more epistemically balanced, scientific approach to clinical research.

Regulatory RNAs and control of epigenetic mechanisms: expectations for cognition and cognitive dysfunction.

The diverse functions of noncoding RNAs (ncRNAs) can influence virtually every aspect of the transcriptional process including epigenetic regulation of genes. In the CNS, regulatory RNA networks and epigenetic mechanisms have broad relevance to gene transcription changes involved in long-term memory formation and cognition. Thus, it is becoming increasingly clear that multiple classes of ncRNAs impact neuronal development, neuroplasticity, and cognition. Currently, a large gap exists in our knowledge of how ncRNAs facilitate epigenetic processes, and how this phenomenon affects cognitive function. In this review, we discuss recent findings highlighting a provocative role for ncRNAs including lncRNAs and piRNAs in the control of epigenetic mechanisms involved in cognitive function. Furthermore, we discuss the putative roles for these ncRNAs in cognitive disorders such as schizophrenia and Alzheimer's disease.

Tunable delivery of bioactive peptides from hydroxyapatite biomaterials and allograft bone using variable-length polyglutamate domains.

Hydroxyapatite (HA) biomaterials and allograft bone are common alternatives to autogenous grafts; however, these materials lack the strong osteoinductive potential of autologous bone. Previous studies have established that polyglutamate domains, which bind selectively to HA, can be engineered onto bioactive peptides as a mechanism for coupling osteoinductive signals onto HA and allograft. In the current investigation, we adapted the polyglutamate approach to tailor delivery of a model collagen-derived peptide, Asp-Gly-Glu-Ala (DGEA), by manipulating the number of glutamates in the HA binding domain. Specifically, DGEA was modified with diglutamate (E2-DGEA), tetraglutamate (E4-DGEA), or heptaglutamate (E7-DGEA), and it was found that initial peptide binding to HA and allograft was significantly enhanced as the number of glutamates increased. We also determined that the rate of release of polyglutamate-DGEA from substrates over a 5-day interval increased proportionally as the number of glutamate residues was decreased. Additionally, we tuned the peptide release rate by creating mixtures of E2-DGEA, E4-DGEA, and E7-DGEA, and observed that release kinetics of the mixtures were distinct from pure solutions of each respective peptide. These collective results suggest that variable-length polyglutamate domains provide an effective mechanism for controlled delivery of osteoregenerative peptides on HA-containing bone graft materials.