Generation of a Dcx-CreERT2 knock-in mouse for genetic manipulation of newborn neurons.

A wide variety of CreERT2 driver lines are available for genetic manipulation of adult-born neurons in the mouse brain. These tools have been instrumental in studying fate potential, migration, circuit integration, and morphology of the stem cells supporting lifelong neurogenesis. Despite a wealth of tools, genetic manipulation of adult-born neurons for circuit and behavioral studies has been limited by poor specificity of many driver lines targeting early progenitor cells and by the inaccessibility of lines selective for later stages of neuronal maturation. We sought to address these limitations by creating a new CreERT2 driver line targeted to the endogenous mouse doublecortin locus as a marker of fate-specified neuroblasts and immature neurons. Our new model places a T2A-CreERT2 cassette immediately downstream of the Dcx coding sequence on the X chromosome, allowing expression of both Dcx and CreERT2 proteins in the endogenous spatiotemporal pattern for this gene. We demonstrate that the new mouse line drives expression of a Cre-dependent reporter throughout the brain in neonatal mice and in known neurogenic niches of adult animals. The line has been deposited with the Jackson Laboratory and should provide an accessible tool for studies targeting fate-restricted neuronal precursors.

EZH2 Methyltransferase Activity Controls Pten Expression and mTOR Signaling during Fear Memory Reconsolidation.

Memory retrieval induces a transient period of increased transcriptional and translational regulation in neurons called reconsolidation, which is regulated by the protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway. However, it is currently unknown how activation of the AKT-mTOR pathway is regulated during the reconsolidation process. Here, we found that in male rats retrieval of a contextual fear memory transiently increased Enhancer of Zeste Homolog 2 (EZH2) levels along with increased histone H3 lysine 27 trimethylation (H3K27me3) levels, which correlated with decreased levels of phosphatase and tensin homolog (PTEN), a potent inhibitor of AKT-mTOR-dependent signaling in the hippocampus. Further experiments found increased H3K27me3 levels and DNA methylation across the Pten promoter and coding regions, indicating transcriptional silencing of the Pten gene. Pten H3K27me3 levels did not change following training or after the retrieval of a remote (old) fear memory, suggesting that this mechanism of Pten repression was specific to the reconsolidation of a new memory. In vivo siRNA-mediated knockdown of Ezh2 in the hippocampus abolished retrieval-induced increases in H3K27me3 and prevented decreases in PTEN levels. Ezh2 knockdown attenuated increases in the phosphorylation of AKT and mTOR following retrieval, which could be restored by simultaneously reducing Pten, suggesting that H3K27me3 regulates AKT-mTOR phosphorylation via repression of Pten Consistent with these results, knockdown of Ezh2 in area CA1 before retrieval impaired memory on later tests. Collectively, these results suggest that EZH2-mediated H3K27me3 plays a critical role in the repression of Pten transcription necessary for AKT-mTOR activation and memory reconsolidation following retrieval.SIGNIFICANCE STATEMENT Understanding how critical translation pathways, like mTOR-mediated protein synthesis, are regulated during the memory storage process is necessary for improving memory impairments. This study tests whether mTOR activation is coupled to epigenetic mechanisms in the hippocampus following the retrieval of a contextual fear memory. Specifically, this study evaluates the role of epigenetic modifications in the form of histone methylation in downstream mTOR translational control during learning-dependent synaptic plasticity in neurons. Considering the broad implications of transcriptional and translational mechanisms in synaptic plasticity, psychiatric, and neurological and neurodegenerative disorders, these data are of interest to the neuroscience community due to the robust and specific regulation of mTOR signaling we found to be dependent on repressive histone methylation.

Activity disruption causes degeneration of entorhinal neurons in a mouse model of Alzheimer's circuit dysfunction.

Neurodegenerative diseases are characterized by selective vulnerability of distinct cell populations; however, the cause for this specificity remains elusive. Here, we show that entorhinal cortex layer 2 (EC2) neurons are unusually vulnerable to prolonged neuronal inactivity compared with neighboring regions of the temporal lobe, and that reelin + stellate cells connecting EC with the hippocampus are preferentially susceptible within the EC2 population. We demonstrate that neuronal death after silencing can be elicited through multiple independent means of activity inhibition, and that preventing synaptic release, either alone or in combination with electrical shunting, is sufficient to elicit silencing-induced degeneration. Finally, we discovered that degeneration following synaptic silencing is governed by competition between active and inactive cells, which is a circuit refinement process traditionally thought to end early in postnatal life. Our data suggests that the developmental window for wholesale circuit plasticity may extend into adulthood for specific brain regions. We speculate that this sustained potential for remodeling by entorhinal neurons may support lifelong memory but renders them vulnerable to prolonged activity changes in disease.

Neurodegenerative conditions cause irreversible damage to the brain and have a devastating impact on quality of life. However, these diseases start gradually, meaning that the entire brain is not affected at once. For example, the initial signs of Alzheimer's disease appear only in specific areas. One of the first brain regions to degenerate in Alzheimer's is the entorhinal cortex. In healthy individuals, entorhinal neurons send electrical signals to the hippocampus, a part of the brain important for memory and learning. During Alzheimer's, hippocampal neurons also die off, leading to 'shrinkage' of this brain region and, ultimately, the memory problems that are a hallmark of the disease. Many neurons in the developing brain require electrical input from other cells to survive ­ in other words, if they do not belong to an 'active circuit', they are eliminated. This is crucial for the connection between the entorhinal cortex and the hippocampus, where the circuit's development and maintenance require carefully controlled electrical activity. Abnormal electrical activity is also an early sign of diseases like Alzheimer's, but how this relates to degeneration is still poorly understood. By investigating these questions, Zhao, Grunke, Wood et al. uncovered a potential relationship between electrical activity and degeneration in the adult brain, long after the circuit between the hippocampus and the entorhinal cortex had matured. Mice were genetically engineered so that their entorhinal cortex would carry a protein designed to silence electrical signaling. The communication between the entorhinal cortex and the hippocampus could therefore be shut off by activating the protein with an injected drug. Remarkably, within just a few days of silencing, cells from the entorhinal cortex started to die off. Zhao, Grunke, Wood et al. went on to show that different silencing methods yielded the same results ­ in other words, the degeneration of cells from the entorhinal cortex was not linked to a particular method. This vulnerability to electrical inactivity was also unique to the entorhinal cortex: when neighboring parts of the brain were silenced, the nerve cells in these areas did not die as readily. Interestingly, in one of their experiments, Zhao, Grunke, Wood et al. found that electrical activity of neighboring nerve cells participated in killing the silenced neurons, suggesting that nerve cells in these brain areas might compete to survive. Overall, this work highlights a direct link between electrical activity and nerve cell degeneration in a part of the brain severely affected by Alzheimer's. In the future, Zhao, Grunke, Wood et al. hope that these results will pave the way to a better understanding of the biological mechanisms underpinning such neurodegenerative diseases.

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.