2010 — 2011 |
Jarome, Timothy Joseph |
F31Activity Code Description: To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
The Role of Proteasome-Dependent Protein Degradation in Fear Memory Reconsolidati @ University of Wisconsin Milwaukee
DESCRIPTION (provided by applicant): Through the proposed research and training I will improve my research skills and continue to investigate neural substrates underlying the complex phenomenon of memory. Specifically, the long-term goal of this project is to understand the contribution of ubiquitin-proteasome mediated protein degradation to the stability of long-term memory in the amygdala following retrieval. The contribution of this molecular process will be examined using two approaches. The first approach is to quantify the rate of protein degradation following memory retrieval using a highly selective GST-protein fusion system, which is an in vitro protein purification technique, and highly specific tissue fractionation procedure, which provides a synaptosomal membrane fraction. The second approach is to manipulate specific molecular processes in the amygdala with infusions of drugs that prevent protein degradation through the ubiquitin-proteasome system, protein synthesis by blocking translation, glutaminergic activity by blocking NMDA receptors, and intracellular signaling by blocking several protein kinases. Four specific aims will be addressed. The first aim is to determine whether protein degradation is increased in the amygdala following the retrieval of context and auditory fear memories. To achieve this aim, fear conditioning will be used to create aversive context- and auditory-based memories and the rate of protein degradation will be quantified in the amygdala following retrieval using the GST-protein fusion system. The second aim, which addresses what the specific synaptic targets of the ubiquitin- proteasome system are following retrieval, will be achieved using the synaptosomal membrane preparation and GST-protein fusion system. The third aim, which addresses whether protein degradation in the amygdala is critical for the reconsolidation of context and auditory fear memories, will be achieved using targeted infusions of drugs that block protein degradation and protein synthesis. The final aim is to determine what molecular mechanisms signal these increases in protein degradation within the amygdala following retrieval. To achieve this aim, infusions of drugs which block NMDA receptor activity and several intracellular signaling cascades will be given prior to retrieval and the rate of protein degradation will be quantified following retrieval using the GST-protein fusion system. Collectively, this set of experiments will answer questions about whether proteasome-dependent protein degradation 1) is involved in postsynaptic density rearrangement following retrieval, 2) is critically involved in amygdala-dependent fear memory reconsolidation, 3) is underlying the requirement for protein synthesis in the reconsolidation process and 4) is being triggered by the same mechanisms which regulate protein synthesis and the "destabilization" of stored memory following retrieval. PUBLIC HEALTH RELEVANCE: Using the Pavlovian fear conditioning paradigm, the proposed experiments outlined in this document will expand the current literature on the physiological underpinnings of memory formation. Enhanced understanding of the biological mechanisms underlying memory is important not only for increasing our basic knowledge of this process but also for the potential translation of research findings to both normal and disordered memory in humans.
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1 |
2012 |
Jarome, Timothy Joseph |
F31Activity Code Description: To provide predoctoral individuals with supervised research training in specified health and health-related areas leading toward the research degree (e.g., Ph.D.). |
The Role of Protein Degradation in Fear Memory Reconsolidation @ University of Wisconsin Milwaukee
Through the proposed research and training I will improve my research skills and continue to investigate neural substrates underlying the complex phenomenon of memory. Specifically, the long-term goal of this project is to understand the contribution of ubiquitin-proteasome mediated protein degradation to the stability of long-term memory in the amygdala following retrieval. The contribution of this molecular process will be examined using two approaches. The first approach is to quantify the rate of protein degradation following memory retrieval using a highly selective GST-protein fusion system, which is an in vitro protein purification technique, and highly specific tissue fractionation procedure, which provides a synaptosomal membrane fraction. The second approach is to manipulate specific molecular processes in the amygdala with infusions of drugs that prevent protein degradation through the ubiquitin-proteasome system, protein synthesis by blocking translation, glutaminergic activity by blocking NMDA receptors, and intracellular signaling by blocking several protein kinases. Four specific aims will be addressed. The first aim is to determine whether protein degradation is increased in the amygdala following the retrieval of context and auditory fear memories. To achieve this aim, fear conditioning will be used to create aversive context- and auditory-based memories and the rate of protein degradation will be quantified in the amygdala following retrieval using the GST-protein fusion system. The second aim, which addresses what the specific synaptic targets of the ubiquitin- proteasome system are following retrieval, will be achieved using the synaptosomal membrane preparation and GST-protein fusion system. The third aim, which addresses whether protein degradation in the amygdala is critical for the reconsolidation of context and auditory fear memories, will be achieved using targeted infusions of drugs that block protein degradation and protein synthesis. The final aim is to determine what molecular mechanisms signal these increases in protein degradation within the amygdala following retrieval. To achieve this aim, infusions of drugs which block NMDA receptor activity and several intracellular signaling cascades will be given prior to retrieval and the rate of protein degradation will be quantified following retrieval using the GST-protein fusion system. Collectively, this set of experiments will answer questions about whether proteasome-dependent protein degradation 1) is involved in postsynaptic density rearrangement following retrieval, 2) is critically involved in amygdala-dependent fear memory reconsolidation, 3) is underlying the requirement for protein synthesis in the reconsolidation process and 4) is being triggered by the same mechanisms which regulate protein synthesis and the destabilization of stored memory following retrieval.
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1 |
2020 |
Jarome, Timothy Joseph Xie, Hehuang [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Exploring Epigenetic Regulation of Memory Extinction @ Virginia Polytechnic Inst and St Univ
TITLE: EXPLORING EPIGENETIC REGULATION OF MEMORY EXTINCTION PROJECT SUMMARY The broad goal of this proposal is to understand how fear memories are extinguished in the brain. Extinction learning, in which continued exposure to cues associated with an aversive event result in reduced responding to these cues, has been proposed as a way to modify or erase fear memories. However, the molecular mechanisms that control the extinction process remain poorly defined, limiting the therapeutic potential of this behavioral process. Recent evidence suggests that extinction learning requires the functions of neuronal activity induced transcription factors including EGR1 and DNA demethylation enzyme TET1 in the medial prefrontal cortex (mPFC) and dorsal hippocampus (DHPC), though it is unknown how these two mechanisms regulate the extinction process. Our recent study indicated that EGR1 recruits TET1 to remove methylation marks on brain DNA during early postnatal development, though whether such a relationship exists during learning-dependent synaptic plasticity in the adult brain remains equivocal. The purpose of this grant is to explore how these two proteins interact to control memory extinction processes with two specific aims. In Aim 1, the investigators will determine the epigenetic roles of EGR1 and TET1 in the mPFC and DHPC during the extinction of fear memory using a combination of behavioral paradigms in genetic knockout mouse models with whole-genome next generation sequencing approaches. In Aim 2, using targeted CRISPR-dCas9 manipulations in the mPFC and DHPC, the investigators will determine the epigenetic role of EGR1-dependent recruitment of TET1 to the memory permissive gene Npas4 during memory extinction. Collectively, the success of this project will provide novel insights into our understanding of the epigenetic role of Egr1/Tet1 and Npas4 genes during extinction consolidation and significantly broaden our understanding of the mechanisms underlying memory extinction, which could potentially lead to new treatment therapeutic strategies for the treatment fear memories associated with a variety of psychiatric disorders.
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0.906 |
2020 |
Gilmartin, Marieke R (co-PI) [⬀] Jarome, Timothy Joseph |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Genome-Wide Analysis of Sex Differences in Cortical Dna Hydroxymethylation During Fear Memory Formation @ Virginia Polytechnic Inst and St Univ
Project Summary/Abstract Post-traumatic stress disorder (PTSD) affects nearly 8% of the population and is more prevalent in women than men. The neurobiological factors that contribute to this sex bias are largely unknown. This project addresses this gap, in accordance with NIMH Strategic Objective 1, by determining how transcriptional regulation of memory-related genes differs in males and females in support of fear memory. Recently our group demonstrated that male and female rats differentially engage signaling mechanisms within the prefrontal cortex (PFC) during the formation of a fear memory, and our preliminary findings point to sex differences in epigenetic modification of prefrontal genes. Currently, the transcriptional regulation of episodic memory within cognitive systems is poorly understood, and the impact of sex on this dynamic process is all but unknown. This represents a significant challenge to developing effective treatment for disorders such as PTSD. The objective of this proposal is to determine how DNA hydroxymethylase TET enzymes functionally regulate fear memory formation and to identify sex-specific methylomic signatures of aversive experience. DNA 5- hydroxymethylation (5-hmC), a major regulator of active (increased) transcription in cells, has been recently implicated in fear memory formation in the brain. However, whether sex differences exist in the engagement of DNA 5-hmC mechanisms during fear memory formation remain equivocal. In our preliminary studies we found that in the rat prefrontal cortex the DNA hydroxymethylase Tet2 increased in females while Tet1 decreased in males following learning in a trace fear conditioning paradigm. The central hypothesis is that TET-mediated DNA 5-hydroxymethylation of sex-specific gene targets in the prefrontal cortex facilitates the formation of fear memory. The approach uses unbiased genome-wide analysis (NIMH Strategy 1.2, Research Priority B) in combination with viral-mediated transcriptional control to test the functional link between TET activity and memory (NIMH Strategy 1.1, Research Priority D; NIMH Strategy 1.2, Research Priority B). Aim 1 will identify the sex-specific gene targets of TET1 and TET2-mediated DNA 5-hmC in the prefrontal cortex following fear learning, using next generation whole genome chromatin immunoprecipitation (ChIP) and hydroxymethylated DNA immunoprecipitation (hmeDIP) sequencing methods. Aim 2 will use cutting-edge CRISPR-dCas9 technology to manipulate the expression of Tet1 and Tet2 in the prefrontal cortex of males and females during trace fear conditioning, which will determine the sex-dependent functional role of cortical TET1 and TET2 in fear memory formation. The proposed research is significant because it is expected to provide a (epi)genomic map of memory formation in a sexually dimorphic brain region, the medial prefrontal cortex, that is needed for understanding and even identifying new genetic biomarkers of sex-biased emotional and cognitive deficits in psychiatric illness.
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0.906 |
2020 — 2021 |
Jarome, Timothy Joseph |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
The Role of Linear Ubiquitination in Memory Formation @ Virginia Polytechnic Inst and St Univ
Project Summary/Abstract The broad goal of this proposal is to understand how memories are formed and stored in the brain. Pavlovian fear conditioning has proven to be a useful paradigm in elucidating the molecular mechanisms underlying memory formation and storage in cells. Indeed, good evidence now exists suggesting that memories formed using this paradigm require dynamic post-translational modification of proteins in neurons. Recently, evidence has emerged demonstrating that protein ubiquitination is critically involved in memory formation in the brain, primarily through targeting proteins for degradation by the proteasome. However, much remains unknown about how other ubiquitin marks which are independent of the proteasome are involved in the memory storage process. In our preliminary studies, we found that in the amygdala fear learning increased the levels of linear ubiquitinated proteins, an atypical ubiquitin modification that is not targeted for degradation by the proteasome complex. Importantly, these increases occurred selectively in the nucleus, but not the cytoplasm or at synapses, and targeted the transcription factor p65, suggesting that linear ubiquitination may be involved in transcriptional control during memory formation. The work in this proposal is designed to answer important questions about whether linear ubiquitination is involved in gene transcription critical for fear memory formation in the amygdala. Using a combination of protein purification methods and mass spectrometry, Aim 1 will identify what proteins are targeted by linear ubiquitination following learning. Additionally, this aim will correlate these identified proteins with next generation RNA-seq data obtained following siRNA-mediated reductions in linear ubiquitination within the amygdala, which will allow an unbiased, whole genome analysis of how this ubiquitin mark is involved in transcriptional processes during the process of memory formation. Aim 2 directly tests the functional role of linear ubiquitination in memory formation by reducing or enhancing this ubiquitin mark in the amygdala via in vivo siRNA or CRISPR-dCas9 manipulations, respectively, and testing memory retention for a contextual fear conditioning task. Collectively, this research will provide critical information regarding how linear ubiquitination is involved in transcriptional regulation during memory formation and whether it is critical for learning-dependent synaptic plasticity. This could provide potentially useful information on how memories are stored in the brain which could have important implications for the treatment of memory impairments associated with a variety of psychiatric disorders.
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0.906 |